Cd19 binding agents and uses thereof

ABSTRACT

This invention, inter alia, relates to CD19 binding agents and methods of using such CD19 binding agents for treating disease.

This application is a divisional of U.S. application Ser. No. 14/743,318filed Jun. 18, 2015, which is a continuation of U.S. application Ser.No. 13/530,074 filed Jun. 21, 2012, now U.S. Pat. No. 9,073,993, whichis a divisional of Ser. No. 13/109,957 filed May 17, 2011, now U.S. Pat.No. 8,242,252, which is a divisional of Ser. No. 12/253,895 filed Oct.17, 2008, now U.S. Pat. No. 7,968,687, which claims the benefit of U.S.Provisional App. No. 60/981,206 filed Oct. 19, 2007; U.S. ProvisionalApp. No. 60/019,214 filed Jan. 4, 2008; and U.S. Provisional App. No.61/080,169 filed Jul. 11, 2008, each of which is incorporated byreference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes a sequence listing as a text file named“510783_SEQLIST.txt” created on Feb. 13, 2018, and containing 73,727bytes, which is incorporated by reference in its entirety for allpurposes.

FIELD

This invention relates to CD19 binding agents comprising a humanizedvariable region and methods of using such CD19 binding agents fortreating disease characterized by expression of CD19 antigen.

BACKGROUND

In humans, B cells can produce an enormous number of antibody molecules.Such antibody production typically ceases (or substantially decreases)when a foreign antigen has been neutralized. Occasionally, however,proliferation of a particular B cell will continue unabated and canresult in a cancer known as a B cell lymphoma. B-cell lymphomas, such asthe B-cell subtype of non-Hodgkin lymphoma, are significant contributorsto cancer mortality. The response of B-cell malignancies to variousforms of treatment is mixed. Despite the medical importance, research inB-cell mediated diseases such as non-Hodgkin lymphoma has produced onlya small number of clinically usable data and conventional approaches totreat such diseases remain tedious and unpleasant and/or have a highrisk of relapse. For example, although high dose chemotherapy as aprimary treatment for high grade non-Hodgkin lymphoma can improveoverall survival, about 50% of the patients still die of this disease.Devesa et al., J. Nat'l Cancer Inst. 79: 701 (1987). Moreover, low-gradenon-Hodgkin lymphoma-like chronic lymphocytic leukemia and mantle celllymphoma are still incurable. This has stimulated the search foralternative strategies like immunotherapy. Antibodies directed againstcell surface molecules defined by CD antigens represent a uniqueopportunity for the development of therapeutic reagents.

The majority of chronic lymphocytic leukemias are of the B-cell lineage.Freedman, Hematol. Oncol. Clin. North Am. 4: 405, 1990. This type ofB-cell malignancy is the most common leukemia in the Western world.Goodman et al., Leukemia and Lymphoma 22: 1, 1996. The natural historyof chronic lymphocytic leukemia falls into several phases. In the earlyphase, chronic lymphocytic leukemia is an indolent disease,characterized by the accumulation of small maturefunctionally-incompetent malignant B-cells having a lengthened lifespan. Eventually, the doubling time of the malignant B-cells decreasesand patients become increasingly symptomatic. While treatment canprovide symptomatic relief, the overall survival of the patients is onlyminimally affected. The late stages of chronic lymphocytic leukemia arecharacterized by significant anemia and/or thrombocytopenia. At thispoint, the median survival is less than two years. Foon et al., AnnalsInt. Medicine 113: 525 (1990).

B cells express cell surface proteins which can be utilized as markersfor differentiation and identification. CD19 is a pan-B cell membraneglycoprotein that is expressed from early stages of pre-B celldevelopment through terminal differentiation, regulating B lymphocytedevelopment and function. Expression of CD19 was identified on mostcancers of lymphoid origin, on the vast majority of Non-Hodgkin lymphoma(NHL) and on leukemias, including Chronic Lymphocytic Leukemia (CLL),Acute Lymphoblastic Leukemia (ALL) and Waldenstrom's Macroglobulinemia(WM). Despite major improvements in the treatment of NHL and CLLpatients, the majority will continue to relapse and salvage regimenswith non-cross resistant compounds are required to improve patientsurvival. A need exists in the art for improved methods of treatment.The present invention addresses this and other needs.

SUMMARY

The invention provides, inter alia, CD19 binding agents and methods ofusing such binding agents. In some aspects, the binding agents comprisethe amino acid sequence(s) of a humanized heavy chain variable regionand/or a humanized light chain variable region and specifically bind tohuman CD19. In some embodiments, the CD19 binding agent is anantigen-binding antibody fragment that specifically binds to human CD19.The antibody fragment can be, for example, a Fab, Fab′, F(ab′)₂, Fvfragment, a diabody, a linear antibody, an scFv, or an scFv-Fc.

In some aspects, the CD19 binding agent has a cytotoxic, cytostaticand/or immunomodulatory effect on CD19-expressing cells. Such an effectcan be mediated, for example, by the depletion or inhibition of theproliferation or differentiation of CD19-expressing cells. In someembodiments, the CD19 binding agent can mediate effector function. Insome embodiments, the CD19 binding agent is conjugated to a therapeuticagent. In other embodiments, the CD19 binding agent is unconjugated,i.e., not conjugated to a therapeutic agent (for example, an anti-CD19naked antibody).

The present invention provides, inter alia, ligand-drug conjugatecompounds wherein the ligand unit is a CD19 binding agent of the presentinvention. The ligand-drug conjugates can be used, for example, to treatan immune disorder or cancer.

Cancers to be treated by the methods of the present invention includeCD19-expressing cancers, including, for example, B-cell lineagemalignancies such as, for example, B cell lymphoma or B cell leukemia,including, but not limited to, non-Hodgkin lymphoma, chronic lymphocyticleukemia, and acute lymphoblastic leukemia.

Also provided are methods for inhibiting the proliferation ordifferentiation of tumor cells expressing CD19. Such methods can includeadministering to the cells a CD19 binding agent (e.g., an anti-CD19 fulllength antibody or antigen-binding fragment thereof that is notconjugated to a therapeutic agent) or a ligand-drug conjugate, whereinthe ligand unit is a CD19 binding agent (e.g., a full length antibody orantigen-binding fragment thereof) conjugated to a cytotoxic, cytostaticand/or therapeutic agent that specifically binds to and can, forexample, inhibit the proliferation or differentiation of cellsexpressing human CD19.

The present invention encompasses methods for inducing the depletion ofB cells, i.e., peripheral B cells, which are associated with an immunedisorder. Such methods can include administering to the cells a CD19binding agent (e.g., an anti-CD19 full length antibody orantigen-binding fragment thereof that is not conjugated to a therapeuticagent) or a ligand-drug conjugate, wherein the ligand unit is a CD19binding agent, (e.g., a full length antibody or antigen-binding fragmentthereof) conjugated to a cytotoxic, cytostatic and/or therapeutic agent.In some embodiments, the immune disorder can be rheumatoid arthritis,systemic lupus erythematosus, multiple sclerosis or inflammatory boweldisease.

In another aspect, pharmaceutical compositions are provided in which thecomposition comprises a CD19 binding agent (e.g., an anti-CD19 fulllength antibody or antigen-binding fragment thereof that is notconjugated to a therapeutic agent) or a ligand-drug conjugate, whereinthe ligand unit is a CD19 binding agent, (e.g., a full length antibodyor antigen-binding fragment thereof) conjugated to a cytotoxic,cytostatic and/or therapeutic agent, and a pharmaceutically acceptableexcipient.

In another aspect, methods of manufacturing ligand-drug conjugatecompounds are provided. In one aspect, a CD19 binding agent isconjugated to a cytotoxic, cytostatic and/or therapeutic agent eitherdirectly or through a linker, as described more fully below.

The present invention may be more fully understood by reference to thefollowing detailed description, non-limiting examples of specificembodiments, and the appended figures and sequence listing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: FACS Results for Binding of CD19 antibodies to non-human primatespleen cells.

FIG. 2: Binding Analysis of heavy and light chain variable regions toCD19.

FIG. 3: Antitumor activity of naked mBU12 antibody and mBU12-mcMMAF onRamos xenograft SCID model. Groups of mice (5/group) were untreated orreceived mBU12 naked antibody (1 mg/kg), mBU12 naked antibody (5 mg/kg),mBU12-mcMMAF4 (1 mg/kg) and mBU12-mcMMAF4 (5 mg/kg) when tumor sizesaveraged approximately 100 mm³. The dose schedule was q4dx3.

FIG. 4: Antitumor activity of anti-CD19 antibody-drug conjugates onRamos tumor model in SCID mice. Groups of mice (5/group) were untreatedor treated with cBU12-mcMMAF8 (3 mg/kg; complete response 1/5 mice),cHD37-mcMMAF8 (3 mg/kg; complete response 5/5 mice), c4g7-mcMMAF8 (3mg/kg; complete response 0/5 mice), or cFMC63-mcMMAF8 (3 mg/kg; completeresponse 5/5 mice) when tumor size averaged approximately 100 mm³. Thedose schedule was q4dx3 iv. In a CR response, the tumor volume is lessthan 13.5 mm³ for three consecutive measurements during the course ofthe study.

FIG. 5: Antitumor activity of anti-CD19 antibody-drug conjugates onRamos tumor model in SCID mice. Groups of mice (10/group) were untreatedor treated with cBU12-mcMMAF8 (3 mg/kg; complete response 6/10 mice),hBU12-mcMMAF8 (3 mg/kg; complete response 10/10 mice; IgG₁ constantregion),), hBU12-mcMMAF8 (3 mg/kg; complete response 6/10 mice; IgG₄constant region),), cHD37-mcMMAF8 (3 mg/kg; complete response 10/10mice), cFMC63-mcMMAF8 (3 mg/kg; complete response 10/10 mice),c4G7-mcMMAF8 (3 mg/kg; complete response 10/10 mice), or cAC10-mcMMAF8(3 mg/kg; complete response 0/10 mice) when tumor size averagedapproximately 100 mm³. The dose schedule was q4dx4 iv.

FIG. 6: Trough levels of cBU12-mcMMAF8 vs cHD37-mcMMAF8.

FIG. 7: Antitumor activity of anti-CD19 antibody-drug conjugates in aRamos tumor model in SCID mice. Groups of mice (10/group) were untreatedor treated with hBU12-mcMMAF8 (3 mg/kg; complete response 10/10 mice),cHD37-mcMMAF8 (3 mg/kg; complete response 10/10 mice), or cAC10-mcMMAF8(3 mg/kg; complete response 0/10 mice) when tumor size averagedapproximately 100 mm³. The dose schedule was q4dx4 iv.

FIG. 8: Antitumor activity of anti-CD19 antibody-drug conjugates inDoHH2 tumor model in SCID mice. Groups of mice (5/group) were untreatedor treated with cAC10-mcMMAF8 (3 mg/kg), cBU12-mcMMAF8 (3 mg/kg),hBU12-mcMMAF8 (3 mg/kg); cHD37-mcMMAF8 (3 mg/kg) or cFMC63-mcMMAF8 (3mg/kg) when tumor size averaged approximately 100 mm³. The dose schedulewas q4dx4, ip.

FIG. 9: Survival assay for SCID mice treated with anti-CD19antibody-drug conjugates in Nalm-6 tumor model. Groups of mice (5/group)were untreated or treated with cAC10-mcMMAF8 (3 mg/kg), cBU12-mcMMAF8 (3mg/kg), hBU12-mcMMAF8 (3 mg/kg); cHD37-mcMMAF8 (3 mg/kg) orcFMC63-mcMMAF8 (3 mg/kg). The dose schedule was q4dx4, iv.

FIG. 10: Trough levels of hBU12-mcMMAF8 (3 mg/kg) vs cHD37-mcMMAF8 (3mg/kg). 5 mice were treated in each group.

FIG. 11: Antitumor activity of hBU12 antibody-drug conjugates in Ramostumor model in SCID mice. Groups of mice (10/group) were untreated ortreated with hBU12-vcMMAE4 (1 mg/kg; complete response 0/10),hBU12-vcMMAE4 (3 mg/kg; complete response 7/10), hBU12-vcMMAF4 (0.3mg/kg; complete response 0/10) hBU12-vcMMAF4 (1 mg/kg; complete response0/10), hBU12-vcMMAF4 (3 mg/kg; complete response 1/10), hBU12-mcMMAF8 (1mg/kg; complete response 0/10), or hBU12-mcMMAF8 (3 mg/kg; completeresponse 10/10). The dose schedule was q4dx4, iv.

FIG. 12: Antitumor activity of anti-CD19 antibody-drug conjugates inDoHH2 tumor model in SCID mice. Groups of mice (1/group) were treatedwith varying 1 mg/kg, 3 mg/kg, or 10 mg/kg of hBU12-vcMMAE4, -mcMMAF4,and -mcMMAF8. The dose schedule was a single dose, ip.

FIG. 13: Ramos cells were cultured with anti-CD19 antibodiescross-linked with a 2-fold excess of goat-anti-mouse ligand drugconjugate (vcMMAF8). Cultures were incubated for 96 hours and labeledwith 50 μM resazurin. Values are the mean±SD of four replicates within asingle experiment.

FIG. 14: CD19 and CD21 expression levels and cytotoxicity ofhBU12-vcMMAE4 and hBU12-mcMMAF4 against ALL, CLL, and NHL tumor celllines grown in culture.

FIG. 15: Internalization kinetics and intracellular trafficking ofhBU12-vcMMAE4 on NHL and ALL tumor cell lines.

FIGS. 16A-16E: Xenograft experiments testing hBU12-vcMMAE4 in models ofNHL. SCID mice were implanted subcutaneously with 5×10⁶ cells of tumorcells in the right flank and treatment was initiated when the averagetumor volume reached 100 mm³. Treatment was intraperitoneally with 1 or3 mg/kg, q4dx4 of hBU12-vcMMAE4. There were 7-10 mice per each treatmentgroup. 16A.) Growth curve of the NHL cell line (Burkitt's lymphoma);16B.) Growth curve of the follicular lymphoma cell lines DOHH2. 16C.)Growth curve of the diffuse large B cell lymphoma (DLBCL) cell lineDLCL2. 16D.) Survival curve of mice implanted with the ALL cell lineRS4; 11 via tail vein. Treatment of mice was initiated on day 7 posttumor implantation at a q4dx4, schedule, intraperitoneally. 16E.)Survival curve of mice implanted with the ALL cell line Nalm-6 via tailvein. Treatment was initiated on day 7 post tumor implantation, with asingle dose of hBU12-vcMMAE4 at the indicated dose, via intraperitonealinjections.

FIGS. 17A-17D: Efficacy of hBU12-vcE in rituximab resistant lymphomas.17A.) SCID mice were implanted with 5×10⁶ of the parental Ramos-P celllines used to generate rituximab resistant tumors. Comparable levels oftumor growth inhibiton were achieved by rituximab (12 mg/kg, q4dx4) andhBU12-vcE (3 mg/kg, q4dx4) 16B.) Tumor growth curve of rituximabresistant Ramos tumors (R-Ramos) treated with hBU12-vcE (1 and 3 mg/kg,IP, q4dx4) or rituximab (12 mg/kg, q4dx4, IP). There was a statisticallysignificant difference in tumor growth delay induced by these compounds17C.) FACS analysis of CD19 and CD20 expression on cells isolated fromRamos-P (sensitive) and R-Ramos (resistant) tumors. Comparableexpression levels for both antigens were identified in both tumors.17D.) Anti-lymphoma effects of hBU12-vcE against subcutaneouslyimplanted, rituximab resistant Raji2R tumors (NHL, Burkitt's lymphoma)treated with 1 and 3 mg/kg, q4dx4 of hBU12-vcE or control conjugate. 9out of 10 durable regressions were observed in hBU12-vcE treated mice,while rituximab (12 mg/kg, q4dx4) did not significantly impact tumorgrowth. There were 5-10 mice per group. A durable response (DR) isdefined as complete absence of palpable tumor during the entireexperiment.

FIG. 18: Activity of hBU12 in disseminated model of ALL. The micereceived a single dose of 10 mg/kg hBU12, hBU12-vcE(4) or hBU12-mcF(4),IP, on day 1 post tumor implanatation. There were 10 mice per group.

FIG. 19: Limited anti-tumor effects of hBU12 in a subcutaneous model ofNHL (SUDHL4). There were 8-10 mice per treatment group. The doseschedule was Q4dx4, IP.

DETAILED DESCRIPTION

The present invention provides, inter alia, CD19 binding agents thatspecifically bind to human CD19. The present inventors have discoveredthat despite the poor efficacy of ligand-drug conjugate compoundscomprising murine or chimeric BU12 antibodies conjugated to a cytotoxicagent, effective ligand-drug conjugate compounds that target human CD19can be developed. Specifically, the present inventors have designedligand-drug conjugate compounds comprising humanized BU12 antibodies asthe ligand unit conjugated to a cytotoxic agent. These ligand-drugconjugate compounds have surprising efficacy given their murine andchimeric counterparts.

In certain aspects, the CD19 binding agents of the present inventioncomprise at least one of the CDR regions of the antibody mBU12. Incertain aspects, the CD19 binding agents comprise all six of the CDRregions of the mBU12 antibody. In some embodiments, the CDR regions haveat least one, at least two, or at least three conservative amino acidsubstitutions of a CDR of antibody mBU12.

In certain aspects, the CD19 binding agents of the present inventioncomprise an antibody heavy chain variable region and/or an antibodylight chain variable region, including derivatives thereof.

In some aspects, the compositions and methods relate to antibodies,including antibody derivatives, that bind to CD19. In certain aspects,the anti-CD19 antibodies and derivatives comprise the amino acidsequence of a humanized heavy chain variable region and/or a humanizedlight chain variable region of antibody BU12, including derivativesthereof. In certain aspects, the anti-CD19 antibodies and derivativescomprise at least one, at least two, at least three, at least four, atleast five, or all six of the CDR regions of antibody mBU12. In someembodiments, the anti-CD19 antibodies include at least oneimmunoglobulin constant region domain, or an entire constant region ofan antibody, such as a human constant region or, optionally, afunctionally active portion thereof. In some embodiments, the antibodyconstant region or domain(s) is of the IgG class. In some embodiments,the antibody constant domain is IgG1, IgG2, or IgG1V1.

In certain aspects, the compositions and methods relate to antibodies,including antibody derivatives, that bind to CD19 and are conjugated tocytotoxic, cytostatic and/or therapeutic agents. In certain embodiments,the antibodies have altered glycosylation patterns.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections whichfollow.

Definitions and Abbreviations

When a trade name is used herein, reference to the trade name alsorefers to the product formulation, the generic drug, and the activepharmaceutical ingredient(s) of the trade name product, unless otherwiseindicated by context.

The terms “CD19 binding agent” and “anti-CD19 binding agent” as usedherein refers to a molecule that specifically binds to CD19. Examplescan include a full length anti-CD19 antibody, a fragment of a fulllength anti-CD19 antibody, or other agent that includes an antibodyheavy and/or light chain variable region, and derivatives thereof.

The terms “specific binding” and “specifically binds” mean that the CD19binding agent will react, in a highly selective manner, with itscorresponding target, CD19 and not with the multitude of other antigens.Typically, the C19 binding agent binds with an affinity of at leastabout 1×10⁻⁷ 10⁻¹, and preferably 10⁻⁸ M to 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or10⁻¹² M and binds to the predetermined antigen with an affinity that isat least two-fold greater than its affinity for binding to anon-specific antigen (e.g., BSA, casein) other than the predeterminedantigen or a closely-related antigen

As used herein, the term “functional,” in the context of a CD19 bindingagent, indicates that the binding agent is capable of specificallybinding to CD19.

The terms “inhibit” or “inhibition of” as used herein means to reduce bya measurable amount, or to prevent entirely.

The term “deplete,” in the context of the effect of a CD19 binding agenton CD19-expressing cells, refers to a reduction in the number of orelimination of the CD19-expressing cells.

“Native antibodies” and “native immunoglobulins” are defined herein asheterotetrameric glycoproteins, typically of about 150,000 daltons,composed of two light (L) chain and two heavy (H) chains. Each lightchain is covalently linked to a heavy chain by a disulfide bond to forma heterodimer. The heterotetramer is formed by covalent disulfidelinkage between the two heavy chains of such heterodimers. Although thelight and heavy chains are linked together by a disulfide bond, thenumber of disulfide linkages between the two heavy chains varies byimmunoglobulin (Ig) isotype. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atthe amino-terminus a variable domain (V_(H)), followed by three or fourconstant domains (C_(H)1, C_(H)2, C_(H)3, and/or C_(H)4, as appropriatefor the antibody type), as well as a hinge (J) region between C_(H)1 andC_(H)2. Each light chain has two domains, an amino-terminal variabledomain (V_(L)) and a carboxy-terminal constant domain (C_(L)). The V_(L)domain associates non-covalently with the V_(H) domain, whereas theC_(L) domain is commonly covalently linked to the C_(H)1 domain via adisulfide bond. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains (see, e.g.,Chothia et al., 1985, J. Mol. Biol. 186:651-663).

The term “hypervariable” refers to certain sequences within the variabledomains of an immunoglobulin that differ extensively in sequence amongantibodies and contain residues that are directly involved in thebinding and specificity of each particular antibody for its specificantigenic determinant. Hypervariability, both in the light chain and theheavy chain variable domains, is concentrated in three segments known ascomplementarity determining regions (CDRs) or hypervariable loops(HVLs). The locations of the CDRs are defined by sequence comparison inKabat et al., 1991, In: Sequences of Proteins of Immunological Interest,5^(th) Ed. Public Health Service, National Institutes of Health,Bethesda, Md., whereas HVLs are structurally defined according to thethree-dimensional structure of the variable domain, as described byChothia and Lesk, 1987, J. Mol. Biol. 196:901-917. As defined by Kabat,CDR-L1 is positioned at about residues 24-34, CDR-L2 at about residues50-56, and CDR-L3 at about residues 89-97 in the light chain variabledomain. CDR-H1 is positioned at about residues 31-35, CDR-H2 at aboutresidues 50-65, and CDR-H3 at about 95-102 in the heavy chain variabledomain.

The three CDRs within each of the heavy and light chains are separatedby framework regions (FR), which contain sequences that tend to be lessvariable. From the amino terminus to the carboxy terminus of the heavyand light chain variable domains, the FRs and CDRs are arranged in theorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The largely β-sheetconfiguration of the FRs brings the CDRs within each of the chains toclose proximity to each other as well as to the CDRs from the otherchain. The resulting conformation contributes to the antigen bindingsite (see, e.g., Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I,pages 647-669), although not all CDR residues are necessarily directlyinvolved in antigen binding.

FR residues and Ig constant domains are typically not directly involvedin antigen binding, but may contribute to antigen binding or mediateantibody effector function. Some FR residues can have a significanteffect on antigen binding in at least three ways: by noncovalentlybinding directly to an epitope, by interacting with one or more CDRresidues, and by affecting the interface between the heavy and lightchains. In some embodiments, the constant domains mediate various Igeffector functions, such as participation of the antibody in antibodydependent cellular cytotoxicity (ADCC), complement dependentcytotoxicity (CDC) and/or antibody dependent cellular phagocytosis(ADCP).

The light chains of vertebrate immunoglobulins are assigned to one oftwo clearly distinct classes, kappa (κ) and lambda (λ), based on theamino acid sequence of the constant domain. By comparison, the heavychains of mammalian immunoglobulins are assigned to one of five majorclasses, according to the sequence of the constant domains: IgA, IgD,IgE, IgG, and IgM. IgG and IgA are further divided into subclasses(isotypes), e.g., IgG₁, IgG₂, IgG₃, and IgG₄; and IgA₁, and IgA₂,respectively. The heavy chain constant domains that correspond to thedifferent classes of immunoglobulins are called α, δ, ε, γ, and μ,respectively. The subunit structures and three-dimensionalconfigurations of the classes of native immunoglobulins are well known.

The terms “antibody”, “anti-CD19 antibody”, “humanized anti-CD19antibody”, and “variant humanized anti-CD19 antibody” are used herein inthe broadest sense and specifically encompass full-length and nativeantibodies, monoclonal antibodies (including full-length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments thereof, such as variabledomains and other portions of antibodies that exhibit a desiredbiological activity (e.g., CD19 binding). The terms “anti-CD19 antibodyfragment”, “humanized anti-CD19 antibody fragment”, and “varianthumanized anti-CD19 antibody fragment” refer to a portion of afull-length anti-CD19 antibody in which a variable region or afunctional capability is retained, for example, specific CD19 epitopebinding. Examples of antibody fragments include, but are not limited to,a Fab, Fab′, F(ab′)2, Fd, Fv, scFv and scFv-Fc fragment, a diabody, alinear antibody, a minibody and a multispecific antibody formed fromantigen-binding antibody fragments. Antibody fragments are specificallyincluded within the definition of “antibody”.

The terms “monoclonal antibody” or “mAb” refer to an antibody obtainedfrom a population of substantially homogeneous antibodies; that is, theindividual antibodies comprising the population are identical except fornaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic determinant, also referred to as an epitope. Themodifier “monoclonal” is indicative of a substantially homogeneouspopulation of antibodies directed to the identical epitope and is not tobe construed as requiring production of the antibody by any particularmethod. Monoclonal antibodies can be made by any technique ormethodology known in the art; for example, the hybridoma method firstdescribed by Köhler et al., 1975, Nature 256:495, or recombinant DNAmethods known in the art (see, e.g., U.S. Pat. No. 4,816,567). Inanother example, monoclonal antibodies also can be isolated from phageantibody libraries, using techniques described in Clackson et al., 1991,Nature 352: 624-628, and Marks et al., 1991, J. Mol. Biol. 222: 581-97.

The term “chimeric” antibody, as used herein, refers to a type ofmonoclonal antibody in which a portion of or the complete amino acidsequence in one or more regions or domains of the heavy and/or lightchain is identical with, homologous to, or a variant of, thecorresponding sequence in a monoclonal antibody from another species orbelonging to another immunoglobulin class or isotype, or from aconsensus sequence. An example of a chimeric antibody is one which has avariable region derived from a non-human monoclonal antibody and a humanIgG immunoglobulin constant region. Chimeric antibodies includefragments of such antibodies, provided that the antibody fragmentexhibits the desired biological activity of its parent antibody, forexample binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567;and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855).Methods for producing chimeric antibodies are known in the art. (See,e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; U.S. Pat.Nos. 5,807,715; 4,816,567; and 4,816,397.)

A “single-chain Fv” or “scFv” antibody fragment is a single chain Fvvariant comprising the V_(H) and V_(L) domains of an antibody in whichthe domains are present in a single polypeptide chain and which iscapable of recognizing and binding antigen. The scFv polypeptideoptionally contains a polypeptide linker positioned between the V_(H)and V_(L) domains that enables the scFv to form a desiredthree-dimensional structure for antigen binding, (see, e.g., PlUckthun,1994, In The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburgand Moore eds., Springer-Verlag, New York, pp. 269-315).

The term “diabody” refers to a small antibody fragment having twoantigen-binding sites. Each fragment contains a heavy chain variabledomain (V_(H)) concatenated to a light chain variable domain (V_(L)) toform a V_(H)-V_(L) or V_(L)-V_(H) polypeptide. By using a linker that istoo short to allow pairing between the two domains on the same chain,the linked V_(H)-V_(L) domains are forced to pair with complementarydomains of another chain, creating two antigen-binding sites. Diabodiesare described more fully, for example, in EP 0 404 097; WO 93/11161; andHollinger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448.

The term “linear antibody” refers to an antibody that has a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) that form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific, as described in Zapata et al., 1995, Protein Eng.8(10):1057-1062.

A “humanized” antibody for the purposes herein is an immunoglobulinamino acid sequence variant or fragment thereof which is capable ofbinding to a predetermined antigen and which comprises a frameworkregion having substantially the amino acid sequence of a humanimmunoglobulin and a CDR having substantially the amino acid sequence ofa non-human immunoglobulin.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are referred to herein as “import” residues, whichare typically taken from an “import” antibody domain, particularly avariable domain. An import residue, sequence, or antibody has a desiredaffinity and/or specificity, or other desirable antibody biologicalactivity as discussed herein.

In general, the humanized antibody will comprise substantially all of atleast one, and sometimes two, variable domains in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence from, e.g., a consensus or germlinesequence. The humanized antibody optionally also will comprise at leasta portion of an immunoglobulin Fc region, typically that of a humanimmunoglobulin. In certain aspects, the antibody will contain both thelight chain variable region as well as the heavy chain variable region.The antibody also may include the C_(H)1, hinge (J), C_(H)2, C_(H)3,and/or C_(H)4 regions of the heavy chain, and the C_(L) region of thelight chain, as appropriate.

The humanized antibody will be selected from any class ofimmunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG₁, IgG₂, IgG₃ and IgG₄, and IgA₁, and IgA₂. The choice ofwhich immunoglobulin class or isotype will depend, in part, on thedesired effector function. For example, the ability of humanimmunoglobulins to mediate CDC and ADCC/ADCP is generally in the orderof IgM≈IgG₁≈IgG₃>IgG₂>IgG₄ and IgG₁≈IgG₃>IgG₂/IgM/IgG₄, respectively.The humanized antibody may comprise sequences from more than one classor isotype, and selecting particular constant domains to optimizedesired effector functions is within the ordinary skill in the art. Thehumanized antibody may or may not have effector function.

The FRs and CDRs of the humanized antibody need not correspond preciselyto the parental sequences, e.g., the import CDR or the consensus FR maybe altered by substitution, insertion or deletion of at least oneresidue so that the CDR or FR residue at that site does not correspondto either the consensus or the import antibody. Typically, such changeswill not be extensive. Usually, at least 75% of the humanized antibodyresidues will correspond to those of the parental FR and CDR sequences,more often 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

A “therapeutic agent” is an agent that exerts a cytotoxic, cytostatic,and/or immunomodulatory effect on cancer cells or activated immunecells. Examples of therapeutic agents include cytotoxic agents,chemotherapeutic agents, cytostatic agents, and immunomodulatory agents.Illustrative therapeutic agents include chemotherapeutic drugs,cytotoxins, immunomodulators, chelators, boron compounds, photoactiveagents, photoactive dyes, steroids, radioisotopes and the like.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer.

A “cytotoxic effect” refers to the depletion, elimination and/or thekilling of a target cell(s). A “cytotoxic agent” refers to an agent thathas a cytotoxic and/or cytostatic effect on a cell. The term is intendedto include radioactive isotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰, and Re¹⁸⁶),chemotherapeutic agents, and toxins such as enzymatically active toxinsof bacterial, fungal, plant, or animal origin, and fragments thereof.

A “cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on acell, thereby inhibiting the growth and/or expansion of a specificsubset of cells.

The term “label” refers to a detectable compound or composition that isconjugated directly or indirectly to a binding agent (e.g., anantibody). The label may itself be detectable (e.g., a radioisotopelabel or a fluorescent label) or, in the case of an enzymatic label, maycatalyze a chemical alteration of a substrate compound or compositionthat is detectable. Labeled CD19 binding agents can be prepared and usedin various applications including in vitro and in vivo diagnostics.Useful labels include diagnostic agents such as contrast agents (such asfor magnetic resonance imaging, computed tomography or ultrasound, e.g.,manganese, iron or gadolinium).

An “isolated” nucleic acid molecule is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe nucleic acid. An isolated nucleic acid molecule is other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from the nucleic acid molecule asit exists in natural cells. However, an isolated nucleic acid moleculeincludes a nucleic acid molecule contained in cells that ordinarilyexpress the antibody where, for example, the nucleic acid molecule is ina chromosomal location different from that of natural cells.

The term “control sequence” refers to a polynucleotide sequencenecessary for expression of an operably linked coding sequence in aparticular host organism. The control sequences suitable for use inprokaryotic cells include, for example, a promoter, operator andribosome binding site sequences. Eukaryotic control sequences include,but are not limited to, promoters, polyadenylation signals, andenhancers. These control sequences can be utilized for expression andproduction of CD19 binding agents in prokaryotic and eukaryotic hostcells.

A nucleic acid sequence is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,a nucleic acid presequence or secretory leader is operably linked to anucleic acid encoding a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide; a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleic acidsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading frame. However, enhancers areoptionally contiguous. Linking can be accomplished, for example, byligation at convenient restriction sites. If such sites do not exist,synthetic oligonucleotide adaptors, linkers or other methods known inthe art can be used.

The term “polypeptide” refers to a polymer of amino acids and itsequivalent and does not refer to a specific length of a product; thus,“peptides” and “proteins” are included within the definition of apolypeptide. Also included within the definition of polypeptides are“antibodies” as defined herein. A “polypeptide region” refers to asegment of a polypeptide, which segment may contain, for example, one ormore domains or motifs (e.g., a polypeptide region of an antibody cancontain, for example, one or more complementarity determining regions(CDRs)). The term “fragment” refers to a portion of a polypeptidetypically having at least 20 contiguous or at least 50 contiguous aminoacids of the polypeptide.

Unless otherwise indicated by context, a “derivative” is a polypeptideor fragment thereof having one or more non-conservative or conservativeamino acid substitutions relative to a second polypeptide (also referredto as a “variant”); or a polypeptide or fragment thereof that ismodified by covalent attachment of a second molecule such as, e.g., byattachment of a heterologous polypeptide, or by glycosylation,acetylation, phosphorylation, and the like. Further included within thedefinition of “derivative” are, for example, polypeptides containing oneor more analogs of an amino acid (e.g., unnatural amino acids and thelike), polypeptides with unsubstituted linkages, as well as othermodifications known in the art, both naturally and non-naturallyoccurring.

An “isolated” polypeptide is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the polypeptide,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. An isolated polypeptide includes an isolatedantibody, or a fragment or derivative thereof.

In certain embodiments, the polypeptide will be purified (1) to greaterthan 95% by weight of polypeptide as determined by the Lowry method, andin other aspects to more than 99% by weight, (2) to a degree sufficientto obtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. “Isolated antibody” includes the antibodyin situ within recombinant cells since at least one component of theantibody's natural environment will not be present.

The term “heterologous,” in the context of a polypeptide, means from adifferent source (e.g., a cell, tissue, organism, or species) ascompared with another polypeptide, so that the two polypeptides aredifferent. Typically, a heterologous polypeptide is from a differentspecies.

In the context of immunoglobulin polypeptides, or fragments thereof, asdefined above, “conservative substitution” means one or more amino acidsubstiutions that do not substantially reduce specific binding (e.g., asmeasured by the K_(D)) of the immunoglobulin polypeptide or fragmentthereof to an antigen (e.g., substitutions that increase binding, thatdo not significantly alter binding, or that reduce binding by no morethan about 40%, typically no more than about 30%, more typically no morethan about 20%, even more typically no more than about 10%, or mosttypically no more than about 5%, as determined by standard bindingassays such as, e.g., ELISA).

The terms “identical” or “percent identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides or amino acid residues that are the same, whencompared and aligned for maximum correspondence. To determine thepercent identity, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoor nucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In some embodiments, the two sequences are the samelength.

The term “substantially identical,” in the context of two nucleic acidsor polypeptides, refers to two or more sequences or subsequences thathave at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98% identity, orat least 99% identity (e.g., as determined using one of the methods setforth infra).

The terms “similarity” or “percent similarity” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are the same or conservatively substituted when compared andaligned for maximum correspondence, as measured using one of the methodsset forth infra. By way of example, a first amino acid sequence can beconsidered similar to a second amino acid sequence when the first aminoacid sequence is at least 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical, or conservativelysubstituted, to the second amino acid sequence when compared to an equalnumber of amino acids as the number contained in the first sequence, orwhen compared to an alignment of polypeptides that has been aligned by,for example, one of the methods set forth infra.

In the context of CD19 binding agents of the present invention, aprotein that has one or more polypeptide regions substantially identicalor substantially similar to one or more antigen-binding regions (e.g., aheavy or light chain variable region, or a heavy or light chain CDR) ofan anti-CD19 antibody retains specific binding to an epitope of CD19recognized by the anti-CD19 antibody, as determined using any of variousstandard immunoassays known in the art or as referred to herein.

The determination of percent identity or percent similarity between twosequences can be accomplished using a mathematical algorithm. Anon-limiting example of a mathematical algorithm utilized for thecomparison of two sequences is the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin andAltschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403-410. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid encoding a protein of interest. BLAST protein searches can beperformed with the XBLAST program, score=50, wordlength=3, to obtainamino acid sequences homologous to a protein of interest. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (id.). When utilizingBLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anothernon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti, 1994, Comput. Appl. Biosci. 10:3-5; and FASTAdescribed in Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA85:2444-8. Within FASTA, ktup is a control option that sets thesensitivity and speed of the search. If ktup=2, similar regions in thetwo sequences being compared are found by looking at pairs of alignedresidues; if ktup=1, single aligned amino acids are examined. ktup canbe set to 2 or 1 for protein sequences, or from 1 to 6 for DNAsequences. The default if ktup is not specified is 2 for proteins and 6for DNA. Alternatively, protein sequence alignment may be carried outusing the CLUSTAL W algorithm, as described by Higgins et al., 1996,Methods Enzymol. 266:383-402.

Optionally, any two antibody sequences can be aligned, for example todetermine percent identity, by using the Kabat numbering system so thateach amino acid in one antibody sequence is aligned with the amino acidin the other sequence that has the same Kabat number. After alignment,if a subject antibody region (e.g., the entire mature variable region ofa heavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

“Effector cell” as used herein refers to a cell that expresses a surfacereceptor for the Fc region of an immunoglobulin (FcR). For example,cells that express surface FcR for IgGs including FcγRIII (CD16), FcγRII(CD32) and FcγRI (CD64) can act as effector cells. Such effector cellsinclude monocytes, macrophages, natural killer (NK) cells, neutrophilsand eosinophils.

The term “antibody effector function(s)” as used herein refers to afunction contributed by an Fc region(s) of an Ig. Such function can beeffected by, for example, binding of an Fc effector region (s) to an Fcreceptor on an immune cell with phagocytic or lytic activity or bybinding of an Fc effector region(s) to components of the complementsystem. The CD19 binding agents of the present invention may or may nothave effector function.

A “disorder”, as used herein, and the terms “CD19-associated disorder”and “CD19-associated disease” refer to any condition that would benefitfrom treatment with a CD19 binding agent described herein. This includeschronic and acute disorders or diseases including those pathologicalconditions that predispose the mammal to the disorder in question.Non-limiting examples or disorders to be treated herein include CD19expressing cancers, including hematological malignancies, benign andmalignant tumors, leukemias and lymphoid malignancies, as well asinflammatory, angiogenic and immunologic disorders. Specific examples ofdisorders are disclosed infra

The terms “treatment” and “therapy”, and the like, as used herein, aremeant to include therapeutic or suppressive measures for a disease ordisorder leading to any clinically desirable or beneficial effect,including, but not limited to, alleviation or relief of one or moresymptoms, regression, slowing or cessation of progression of the diseaseor disorder associated with CD19 expression, such as a cancer. Forexample, treatment can include a decrease or elimination of a clinicalor diagnostic symptom of a CD19-expressing disorder after the onset ofthe clinical or diagnostic symptom by administration of an anti-CD19antibody or other CD19 binding agent to a subject. Treatment can beevidenced as a decrease in the severity of a symptom, the number ofsymptoms, or frequency of relapse.

Except when noted, the terms “subject” or “patient” are usedinterchangeably and refer to mammals such as human patients andnon-human primates, as well as experimental animals such as rabbits,dogs, cats, rats, mice, and other animals. Accordingly, the term“subject” or “patient” as used herein means any mammalian patient orsubject to which the CD19 binding agents of the invention can beadministered. Subjects of the present invention include those that havebeen diagnosed with a CD19 expressing cancer, including, for example, Bcell lymphoma or B cell leukemia, including, but not limited to,non-Hodgkin lymphoma, chronic lymphocytic leukemia, and acutelymphoblastic leukemia. In certain embodiments, the subject will have arefractory or relapsed CD19 expressing cancer

A subject with a refractory CD19 expressing cancer is a subject who doesnot respond to therapy, i.e., the subject continues to experiencedisease progresssion despite therapy.

A subject with a relapsed CD19 expressing cancer is a subject who hasresponded to the therapy at one point, but has had a reoccurence orfurther progression of disease following the response.

The term “effective amount” refers to the amount of a CD19 binding agentor ligand-drug conjugate that is sufficient to inhibit the occurrence orameliorate one or more clinical or diagnostic symptoms of aCD19-associated disorder in a subject. An effective amount of an agentis administered according to the methods described herein in an“effective regimen.” The term “effective regimen” refers to acombination of amount of the agent and dosage frequency adequate toaccomplish treatment or prevention of a CD19-associated disorder.

The term “pharmaceutically acceptable” as used herein refers to thosecompounds, materials, compositions, and/or dosage forms that are, withinthe scope of sound medical judgment, suitable for contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problems or complicationscommensurate with a reasonable benefit/risk ratio. The term“pharmaceutically compatible ingredient” refers to a pharmaceuticallyacceptable diluent, adjuvant, excipient, or vehicle with which a CD19binding agent or a ligand-drug conjugate compound is administered.

The term “pharmaceutically compatible ingredient” refers to apharmaceutically acceptable diluent, adjuvant, excipient, or vehiclewith which a CD19 binding agent is administered.

The term “compound” refers to and encompasses the chemical compounditself as well as, whether explicitly stated or not, and unless thecontext makes clear that the following are to be excluded: amorphous andcrystalline forms of the compound, including polymorphic forms, wherethese forms may be part of a mixture or in isolation; free acid and freebase forms of the compound, which are typically the forms shown in thestructures provided herein; isomers of the compound, which refers tooptical isomers, and tautomeric isomers, where optical isomers includeenantiomers and diastereomers, chiral isomers and non-chiral isomers,and the optical isomers include isolated optical isomers as well asmixtures of optical isomers including racemic and non-racemic mixtures;where an isomer may be in isolated form or in a mixture with one or moreother isomers; isotopes of the compound, including deuterium- andtritium-containing compounds, and including compounds containingradioisotopes, including therapeutically- and diagnostically-effectiveradioisotopes; multimeric forms of the compound, including dimeric,trimeric, etc. forms; salts of the compound, preferably pharmaceuticallyacceptable salts, including acid addition salts and base addition salts,including salts having organic counterions and inorganic counterions,and including zwitterionic forms, where if a compound is associated withtwo or more counterions, the two or more counterions may be the same ordifferent; and solvates of the compound, including hemisolvates,monosolvates, disolvates, etc., including organic solvates and inorganicsolvates, said inorganic solvates including hydrates; where if acompound is associated with two or more solvent molecules, the two ormore solvent molecules may be the same or different. In some instances,reference made herein to a compound of the invention will include anexplicit reference to one or of the above forms, e.g., salts and/orsolvates, however, this reference is for emphasis only, and is not to beconstrued as excluding other of the above forms as identified above.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali or organic salts ofacidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For example,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like. These physiologically acceptable salts are prepared bymethods known in the art, e.g., by dissolving the free amine bases withan excess of the acid in aqueous alcohol, or neutralizing a freecarboxylic acid with an alkali metal base such as a hydroxide, or withan amine

Unless otherwise noted, the term “alkyl” refers to a saturated straightor branched hydrocarbon having from about 1 to about 20 carbon atoms(and all combinations and subcombinations of ranges and specific numbersof carbon atoms therein), with from about 1 to about 8 carbon atomsbeing preferred. Examples of alkyl groups are methyl, ethyl, n-propyl,isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, n-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl,n-nonyl, n-decyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl,1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.

Alkyl groups, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 3 groups(and any additional substituents selected from halogen), including, butnot limited to, halogen, optionally substituted —O—(C₁-C₈ alkyl),optionally substituted —O—(C₂-C₈ alkenyl), optionally substituted—O—(C₂-C₈ alkynyl), optionally substituted aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′,—S(O)₂R′, —S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, whereeach R′ is independently selected from H, optionally substituted —C₁-C₈alkyl, optionally substituted —C₂-C₈ alkenyl, optionally substituted—C₂-C₈ alkynyl, or optionally substituted aryl, and wherein saidoptionally substituted O—(C₁-C₈ alkyl), optionally substituted —O—(C₂-C₈alkenyl), optionally substituted —O—(C₂-C₈ alkynyl), optionallysubstituted aryl, optionally substituted C₁-C₈ alkyl, optionallysubstituted —C₂-C₈ alkenyl, and optionally substituted —C₂-C₈ alkynylgroups can be optionally further substituted with one or more groupsincluding, but not limited to, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Unless otherwise noted, the terms “alkenyl” and “alkynyl” refer tostraight and branched carbon chains having from about 2 to about 20carbon atoms (and all combinations and subcombinations of ranges andspecific numbers of carbon atoms therein), with from about 2 to about 8carbon atoms being preferred. An alkenyl chain has at least one doublebond in the chain and an alkynyl chain has at least one triple bond inthe chain. Examples of alkenyl groups include, but are not limited to,ethylene or vinyl, allyl, -1-butenyl, -2-butenyl, -isobutylenyl,-1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, and-2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but are notlimited to, acetylenic, propargyl, acetylenyl, propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1 butynyl.

Alkenyl and alkynyl groups, whether alone or as part of another group,can be optionally substituted with one or more groups, preferably 1 to 3groups (and any additional substituents selected from halogen),including but not limited halogen, optionally substituted —O—(C₁-C₈alkyl), optionally substituted —O—(C₂-C₈ alkenyl), optionallysubstituted —O—(C₂-C₈ alkynyl), optionally substituted aryl, —C(O)R′,—OC(O)R′, —C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′,—SO₃R′, —S(O)₂R′, —S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN,where each R′ is independently selected from H, optionally substituted—C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkyenl, optionallysubstituted —C₂-C₈ alkynyl, or optionally substituted aryl and whereinsaid optionally substituted —O—(C₁-C₈ alkyl), optionally substituted—O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈ alkynyl),optionally substituted aryl, optionally substituted C₁-C₈ alkyl,optionally substituted —C₂-C₈ alkenyl, and optionally substituted —C₂-C₈alkynyl groups can be optionally further substituted with one or moresubstituents including, but not limited to, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂,—C(O)NHR″, —C(O)N(R″)₂—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH,—N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ is independentlyselected from H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Unless otherwise noted, the term “alkylene” refers to a saturatedbranched or straight chain hydrocarbon radical having from about 1 toabout 20 carbon atoms (and all combinations and subcombinations ofranges and specific numbers of carbon atoms therein), with from about 1to about 8 carbon atoms being preferred and having two monovalentradical centers derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent alkane. Typical alkylenesinclude, but are not limited to, methylene, ethylene, propylene,butylene, pentylene, hexylene, heptylene, ocytylene, nonylene, decalene,1,4-cyclohexylene, and the like. Alkylene groups, whether alone or aspart of another group, can be optionally substituted with one or moregroups, preferably 1 to 3 groups (and any additional substituentsselected from halogen), including, but not limited to, halogen,optionally substituted —O—(C₁-C₈ alkyl), optionally substituted—O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈ alkynyl),optionally substituted aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from H, optionally substituted —C₁-C₈ alkyl, optionallysubstituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈ alkynyl, oroptionally substituted aryl and wherein said optionally substitutedO—(C₁-C₈ alkyl), optionally substituted —O—(C₂-C₈ alkenyl), optionallysubstituted —O—(C₂-C₈ alkynyl), optionally substituted aryl, optionallysubstituted C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkenyl, andoptionally substituted —C₂-C₈ alkynyl groups can be further optionallysubstituted with one or more substituents including, but not limited to,C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl),—O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″,—C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂—NHC(O)R″, —SR″, —SO₃R″,—S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN, where eachR″ is independently selected from H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl,—C₂-C₈ alkynyl, or aryl.

Unless otherwise noted, the term “alkenylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon doublebond. Exemplary alkenylene groups include, for example, ethenylene(—C_(H)═C_(H)—) and propenylene (—C_(H)═CHCH₂—).

Unless otherwise noted, the term “alkynylene” refers to an optionallysubstituted alkylene group containing at least one carbon-carbon triplebond. Exemplary alkynylene groups include, for example, acetylene(—C≡C—), propargyl (—CH₂C≡C—), and 4-pentynyl (—CH₂CH₂CH₂C≡CH—).

Unless otherwise noted, the term “aryl” refers to a monovalent aromatichydrocarbon radical of 6-20 carbon atoms (and all combinations andsubcombinations of ranges and specific numbers of carbon atoms therein)derived by the removal of one hydrogen atom from a single carbon atom ofa parent aromatic ring system. Some aryl groups are represented in theexemplary structures as “Ar”. Typical aryl groups include, but are notlimited to, radicals derived from benzene, substituted benzene, phenyl,naphthalene, anthracene, biphenyl, and the like.

An aryl group, whether alone or as part of another group, can beoptionally substituted with one or more, preferably 1 to 5, or even 1 to2 groups including, but not limited to, halogen, optionally substituted—C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkenyl, optionallysubstituted —C₂-C₈ alkynyl, optionally substituted —O—(C₁-C₈ alkyl),optionally substituted —O—(C₂-C₈ alkenyl), optionally substituted—O—(C₂-C₈ alkynyl), optionally substituted aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′,—S(O)₂R′, —S(O)R′, —OH, —NO₂, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, whereeach R′ is independently selected from H, optionally substituted —C₁-C₈alkyl, optionally substituted —C₂-C₈ alkenyl, optionally substituted—C₂-C₈ alkynyl, or optionally substituted aryl and wherein saidoptionally substituted C₁-C₈ alkyl, optionally substituted —C₂-C₈alkenyl, optionally substituted —C₂-C₈ alkynyl, optionally substituted—O—(C₁-C₈ alkyl), optionally substituted —O—(C₂-C₈ alkenyl), optionallysubstituted —O—(C₂-C₈ alkynyl), and optionally substituted aryl groupscan be further optionally substituted with one or more substituentsincluding, but not limited to, C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO_(3R)″, —S(O)₂R″, —S(O)R″, —OH, —N₃,—NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ is independently selectedfrom H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Unless otherwise noted, the term “arylene” refers to an optionallysubstituted aryl group which is divalent (i.e., derived by the removalof two hydrogen atoms from the same or two different carbon atoms of aparent aromatic ring system) and can be in the ortho, meta, or paraconfigurations as shown in the following structures with phenyl as theexemplary aryl group:

Typical “—(C₁-C₈ alkylene)aryl,” “—(C₂-C₈ alkenylene)aryl, “and —(C₂-C₈alkynylene)aryl groups include, but are not limited to, benzyl,2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl,2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl,2-naphthophenylethan-1-yl and the like.

Unless otherwise noted, the term “heterocycle,” refers to a monocyclic,bicyclic, or polycyclic ring system having from 3 to 14 ring atoms (alsoreferred to as ring members) wherein at least one ring atom in at leastone ring is a heteroatom selected from N, O, P, or S (and allcombinations and subcombinations of ranges and specific numbers ofcarbon atoms and heteroatoms therein). The heterocycle can have from 1to 4 ring heteroatoms independently selected from N, O, P, or S. One ormore N, C, or S atoms in a heterocycle can be oxidized. A monocylicheterocycle preferably has 3 to 7 ring members (e.g., 2 to 6 carbonatoms and 1 to 3 heteroatoms independently selected from N, O, P, or S),and a bicyclic heterocycle preferably has 5 to 10 ring members (e.g., 4to 9 carbon atoms and 1 to 3 heteroatoms independently selected from N,O, P, or S). The ring that includes the heteroatom can be aromatic ornon-aromatic. Unless otherwise noted, the heterocycle is attached to itspendant group at any heteroatom or carbon atom that results in a stablestructure.

Heterocycles are described in Paquette, “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.82:5566 (1960).

Unless otherwise noted, the term “heterocyclo” refers to an optionallysubstituted heterocycle group as defined above that is divalent (i.e.,derived by the removal of two hydrogen atoms from the same or twodifferent carbon atoms of a parent heterocyclic ring system).

Examples of “heterocycle” groups include by way of example and notlimitation pyridyl, dihydropyridyl, tetrahydropyridyl (piperidyl),thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl,imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl,indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, piperidinyl,4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl,tetrahydrofuranyl, bis-tetrahydrofuranyl, tetrahydropyranyl,bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, octahydroisoquinolinyl, azocinyl, triazinyl,6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl,pyranyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl,2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl,indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl. Preferred “heterocycle” groups include, but are notlimited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl,coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl, thiazolyl,imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl,pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl andtetrazolyl.

A heterocycle group, whether alone or as part of another group, can beoptionally substituted with one or more groups, preferably 1 to 2groups, including but not limited to, optionally substituted —C₁-C₈alkyl, optionally substituted —C₂-C₈ alkenyl, optionally substituted—C₂-C₈ alkynyl, halogen, optionally substituted —O—(C₁-C₈ alkyl),optionally substituted —O—(C₂-C₈ alkenyl), optionally substituted—O—(C₂-C₈ alkynyl), optionally substituted -aryl, —C(O)R′, —OC(O)R′,—C(O)OR′, —C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′,—S(O)₂R′, —S(O)R′, —OH, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where eachR′ is independently selected from H, optionally substituted —C₁-C₈alkyl, optionally substituted —C₂-C₈ alkenyl, optionally substituted—C₂-C₈ alkynyl, or optionally substituted aryl and wherein saidoptionally substituted O—(C₁-C₈ alkyl), optionally substituted —O—(C₂-C₈alkenyl), optionally substituted —O—(C₂-C₈ alkynyl), optionallysubstituted —C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkenyl,optionally substituted —C₂-C₈ alkynyl, and optionally substituted arylgroups can be further optionally substituted with one or moresubstituents including, but not limited to, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl),—O—(C₂-C₈ alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂,—C(O)NHR″, —C(O)N(R″)₂—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH,—N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN, where each R″ is independentlyselected from H, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

By way of example and not limitation, carbon-bonded heterocycles can bebonded at the following positions: position 2, 3, 4, 5, or 6 of apyridine; position 3, 4, 5, or 6 of a pyridazine; position 2, 4, 5, or 6of a pyrimidine; position 2, 3, 5, or 6 of a pyrazine; position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole orthiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole;position 2 or 3 of an aziridine; position 2, 3, or 4 of an azetidine;position 2, 3, 4, 5, 6, 7, or 8 of a quinoline; or position 1, 3, 4, 5,6, 7, or 8 of an isoquinoline. Still more typically, carbon bondedheterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl,6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl,2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl,3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, or 1H-indazole;position 2 of a isoindole, or isoindoline; position 4 of a morpholine;and position 9 of a carbazole, or β-carboline. Still more typically,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

Unless otherwise noted, the term “carbocycle,” refers to a saturated orunsaturated non-aromatic monocyclic, bicyclic, or polycyclic ring systemhaving from 3 to 14 ring atoms (and all combinations and subcombinationsof ranges and specific numbers of carbon atoms therein) wherein all ofthe ring atoms are carbon atoms. Monocyclic carbocycles preferably have3 to 6 ring atoms, still more preferably 5 or 6 ring atoms. Bicycliccarbocycles preferably have 7 to 12 ring atoms, e.g., arranged as abicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atomsarranged as a bicyclo [5,6] or [6,6] system. The term “carbocycle”includes, for example, a monocyclic carbocycle ring fused to an arylring (e.g., a monocyclic carbocycle ring fused to a benzene ring).Carbocyles preferably have 3 to 8 carbon ring atoms.

Carbocycle groups, whether alone or as part of another group, can beoptionally substituted with, for example, one or more groups, preferably1 or 2 groups (and any additional substituents selected from halogen),including, but not limited to, halogen, optionally substituted C₁-C₈alkyl, optionally substituted —C₂-C₈ alkenyl, optionally substituted—C₂-C₈ alkynyl, optionally substituted —O—(C₁-C₈ alkyl), optionallysubstituted —O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈alkynyl), optionally substituted aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ isindependently selected from H, optionally substituted —C₁-C₈ alkyl,optionally substituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈alkynyl, or optionally substituted aryl and wherein said optionallysubstituted —C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkenyl,optionally substituted —C₂-C₈ alkynyl, optionally substituted —O—(C₁-C₈alkyl), optionally substituted —O—(C₂-C₈ alkenyl), optionallysubstituted —O—(C₂-C₈ alkynyl), and optionally substituted aryl groupscan be further optionally substituted with one or more substituentsincluding, but not limited to, C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈alkynyl, halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈alkynyl), -aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂, —NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl.

Examples of monocyclic carbocylic substituents include cyclopropyl,cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl,1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl,1-cyclohex-3-enyl, cycloheptyl, and cyclooctyl. -1,3-cyclohexadienyl,-1,4-cyclohexadienyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl,and -cyclooctadienyl.

A “carbocyclo,” whether used alone or as part of another group, refersto an optionally substituted carbocycle group as defined above that isdivalent (i.e., derived by the removal of two hydrogen atoms from thesame or two different carbon atoms of a parent carbocyclic ring system).

When any variable occurs more than one time in any constituent or in anyformula, its definition in each occurrence is independent of itsdefinition at every other. Combinations of substituents and/or variablesare permissible only if such combinations result in stable compounds.

Unless otherwise indicated by context, a hyphen (-) designates the pointof attachment to the pendant molecule. Accordingly, the term “—(C₁-C₈alkylene)aryl” or “—C₁-C₈ alkylene(aryl)” refers to a C₁-C₈ alkyleneradical as defined herein wherein the alkylene radical is attached tothe pendant molecule at any of the carbon atoms of the alkylene radicaland one of the hydrogen atom bonded to a carbon atom of the alkyleneradical is replaced with an aryl radical as defined herein.

When a particular group is “substituted”, that group may have one ormore substituents, preferably from one to five substituents, morepreferably from one to three substituents, most preferably from one totwo substituents, independently selected from the list of substituents.The group can, however, generally have any number of substituentsselected from halogen. Groups that are substituted are so indicated.

It is intended that the definition of any substituent or variable at aparticular location in a molecule be independent of its definitionselsewhere in that molecule. It is understood that substituents andsubstitution patterns on the compounds of this invention can be selectedby one of ordinary skill in the art to provide compounds that arechemically stable and that can be readily synthesized by techniquesknown in the art as well as those methods set forth herein.

Protective groups as used herein refer to groups which selectivelyblock, either temporarily or permanently, one reactive site in amultifunctional compound. Suitable hydroxy-protecting groups for use inthe present invention can be administered to a subject in the context ofthe present invention and may or may not need to be cleaved from theparent compound after administration to a subject in order for thecompound to be active. Cleavage is through normal metabolic processeswithin the body. Hydroxy protecting groups are well known in the art,see, Protective Groups in Organic Synthesis by T. W. Greene and P. G. M.Wuts (John Wiley & sons, 3^(rd) Edition) incorporated herein byreference in its entirety and for all purposes and include, for example,ether (e.g., alkyl ethers and silyl ethers including, for example,dialkylsilylether, trialkylsilylether, dialkylalkoxysilylether), ester,carbonate, carbamates, sulfonate, and phosphate protecting groups.Examples of hydroxy protecting groups include, but are not limited to,methyl ether; methoxymethyl ether, methylthiomethyl ether,(phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl ether,p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl ether,o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl ether,guaiacolmethyl ether, t-butoxymethyl ether, 4-pentenyloxymethyl ether,siloxymethyl ether, 2-methoxyethoxymethyl ether,2,2,2-trichloroethoxymethyl ether, bis(2-chloroethoxy)methyl ether,2-(trimethylsilyl)ethoxymethyl ether, menthoxymethyl ether,tetrahydropyranyl ether, 1-methoxycylcohexyl ether,4-methoxytetrahydrothiopyranyl ether, 4-methoxytetrahydrothiopyranylether S,S-Dioxide, 1-[(2-choro-4-methyl)phenyl]-4-methoxypiperidin-4-ylether, 1-(2-fluorophneyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-ylether, tetrahydrofuranyl ether, tetrahydrothiofuranyl ether; substitutedethyl ethers such as 1-ethoxyethyl ether, 1-(2-chloroethoxy)ethyl ether,1-[2-(trimethylsilyl)ethoxy]ethyl ether, 1-methyl-1-methoxyethyl ether,1-methyl-1-benzyloxyethyl ether, 1-methyl-1-benzyloxy-2-fluoroethylether, 1-mehtyl-1phenoxyethyl ether, 2-trimethylsilyl ether, t-butylether, allyl ether, propargyl ethers, p-chlorophenyl ether,p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl ether,tripropylsilylether, dimethylisopropylsilyl ether, diethylisopropylsilylether, dimethylhexylsilyl ether, t-butyldimethylsilyl ether,diphenylmethylsilyl ether, benzoylformate ester, acetate ester,chloroacetate ester, dichloroacetate ester, trichloroacetate ester,trifluoroacetate ester, methoxyacetate ester, triphneylmethoxyacetateester, phenylacetate ester, benzoate ester, alkyl methyl carbonate,alkyl 9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl2,2,2,-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethylcarbonate, alkylsulfonate, methanesulfonate, benzylsulfonate, tosylate,methylene acetal, ethylidene acetal, and t-butylmethylidene ketal.Preferred protecting groups are represented by the formulas —R^(a),—Si(R^(a))(R^(a))(R^(a)), —C(O)R^(a), —C(O)OR^(a), —C(O)NH(R^(a)),—S(O)₂R^(a), —S(O)₂OH, P(O)(OH)₂, and —P(O)(OH)OR^(a), wherein R^(a) isC₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, —C₁-C₂₀alkylene(carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle) wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene radicals whether alone or as part of anothergroup are optionally substituted with one or more groups independentlyselected from A1, said carbocycle radicals whether alone or as part ofanother group are optionally substituted with one or more groupsindependently selected from A2, said aryl radicals whether alone or aspart of another group are optionally substituted with one or more groupsindependently selected from A3, and said heterocycle radicals whetheralone or as part of another group are optionally substituted with one ormore groups independently selected from A4. A1, A2, A3, and A4 are asdefined herein.

The abbreviation “AFP” refers todimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine(see Formula XVI infra).

The abbreviation “MMAE” refers to monomethyl auristatin E (see FormulaXI infra).

The abbreviation “AEB” refers to an ester produced by reactingauristatin E with paraacetyl benzoic acid (see Formula XX infra)

The abbreviation “AEVB” refers to an ester produced by reactingauristatin E with benzoylvaleric acid (see Formula XXI infra).

The abbreviation “MMAF” refers todovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula IVIVinfra).

CD19 Binding Agents

The methods described herein encompass the use of CD19 binding agentsand ligand-drug conjugate compounds wherein the ligand unit is ananti-CD19 binding agent that specifically binds to CD19. The CD19binding agent can be, for example, an anti-CD19 antibody, an anti-CD19antigen-binding fragment, or other CD19 binding agent comprising theamino acid sequence of a humanized antibody heavy and/or light chainvariable region, or derivative thereof.

In certain aspects, the anti-CD19 binding agents of the presentinvention include a heavy and/or light chain variable domain, the heavyand light chain variable domains each have (a) a set of three CDRsidentical or substantially identical to the corresponding CDRs of mAbmBU12, and (b) a set of four variable region framework regions identicalor substantially identical to framework regions from a humanimmunoglobulin.

The present invention encompasses embodiments wherein the frameworkregions chosen for the heavy chain variable region of the CD19 bindingagents of the present invention are the human germline V_(H) exonsV_(H)2-70 or V_(H)4-31 and the human germline J_(H)4 exon for thehumanized FR4 sequence. In some embodiments, the human germline J_(H)1,J_(H)2, J_(H)3, J_(H)5, or J_(H)6 exon is used in place of the humangermline J_(H)4 exon for the humanized FR4 sequence.

The present invention encompasses embodiments wherein the frameworkregions chosen for the light chain variable region of the CD19 bindingagents of the present invention are the human germline V_(L) exonsV_(L)-L6 or V_(L)A10 and the human germline J_(k)2 exon for thehumanized FR4 sequence. In some embodiments, the human germline J_(k)1,J_(k)3, J_(k)4, or J_(k)5 exon is used in place of the human germlineJ_(k)2 exon for the humanized FR4 sequence.

The present invention encompasses embodiments wherein mouse donorresidues are reintroduced into the sequence of the framework region ofthe CD19 binding agents. Such residues can include, for example,reintroduction of the mouse donor residue at one or more of positions75, 79, 81, 82, 82A, 82B, 82C and 89, according to the Kabat numberingsystem, of the V_(H)2-70/J_(H)4 germline, positions 24, 27, 29, 71, 75,78, 79, and 89, according to the Kabat numbering system, of theV_(H)4-31/J_(H)4 germline, positions 2, 40, 41, 42, 69, 70, 71, 72, and83, according to the Kabat numbering system, of the V_(L)-L6/J_(k)2germline, and positions 2 and 71, according to the Kabat numberingsystem, of the V_(L)A10/J_(k)2 germline. Additional mouse donor residuesat alternate positions can be reintroduced into the sequences.

The present invention emcompasses embodiment wherein the CD19 bindingagents described herein have amino acid sequence modification(s) in theacceptor human germline exon in addition to the reintroduction of mousedonor residues as well as amino acid sequence modification(s) in thehypervariable regions. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the antibody.Amino acid sequence variants of the CD19 binding agents are prepared byintroducing appropriate nucleotide changes into the antibody nucleicacid, or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions of, residueswithin the amino acid sequences of the antibody. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics. Substitutions may be conservative or non-conservativesubstitutions. The amino acid changes also may alter post-translationalprocesses of the antibody, such as changing the number or position ofglycosylation sites.

A useful method for identification of certain residues or regions of theCD19 binding agent that are favored locations for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and WellsScience, 244:1081-1085 (1989). Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed variants arescreened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions as well as intrasequence insertions of single or multiple aminoacid residues. Another type of variant is an amino acid substitutionvariant. These variants have at least one amino acid residue in theantibody molecule replaced by a different residue. The sites of greatestinterest for substitutional mutagenesis include the hypervariableregions, but FR alterations are also contemplated.

Substantial modifications in the biological properties of the CD19binding agent are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.Naturally-occurring residues are divided into groups based on commonside-chain properties:

(1) hydrophobic: met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Conservative substitutions will entailexchanging members of the same class.

One type of substitutional variant involves substituting one or morehypervariable region residues. In some embodiments, the resultingvariant(s) selected for further development will have improvedbiological properties relative to the parent binding agent from whichthey are generated. A convenient way for generating such substitutionalvariants involves affinity maturation using phage display. Briefly,several hypervariable region sites (e.g., 6-7 sites) are mutated togenerate all possible amino substitutions at each site. The variantsthus generated are displayed in a monovalent fashion from filamentousphage particles as fusions to the gene III product of M13 packagedwithin each particle. The phage-displayed variants are then screened fortheir biological activity (e.g., binding affinity) as herein disclosed.In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the binding agent and the antigen. Such contact residuesand neighboring residues are candidates for substitution according tothe techniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andbinding agents with superior properties in one or more relevant assaysmay be selected for further development.

The antibodies or derivatives thereof or other binding agents can havemodifications (e.g., substitutions, deletions or additions) in aminoacid residues that interact with Fcγ receptors. In particular,antibodies or derivatives thereof or other binding agents includeantibodies or derivatives thereof or other binding agents havingmodifications in amino acid residues identified as involved in thebinding interaction between the Fc domain and one or more Fcγ receptors(see infra), as well as antibodies or derivatives thereof or otherbinding agents having modifications in amino acid residues identified asinvolved in the interaction between the anti-Fc domain and the FcRnreceptor (see, e.g., International Publication No. WO 97/34631, which isincorporated herein by reference in its entirety).

In some embodiments, the binding of a target binding agent to one ormore Fcγ receptors can be impaired using one or more antibodyengineering approaches known in the art. In some embodiments, thebinding of a target binding agent to one or more Fcγ receptors can beimpaired by reducing the target binding agent's effector functions usingone or more antibody engineering approaches known in the art.Illustrative, non-limiting examples for such approaches are providedbelow.

Fcγ receptor binding is mediated through the interaction of a region ofan antibody with an Fc gamma (Fcγ) receptor (FcγR). The Fc region ordomain refers to the region(s) of an antibody constant region (e.g.,IgG1, IgG2, IgG3, or IgG4) that is involved in the binding interactionof the Fc region to one or more Fcγ receptors (e.g., FcγRI (CD64),FcγRIIb (CD32b) or FcγRIIIa (CD16). Both the glycosylation status andprimary amino acid sequence of the IgG Fc region have functional effectson the Fc region-FcγR interaction.

Substitution of particular amino acid positions in the Fc region of IgGisotype constant regions are known to have functional effects on theability of an antibody to bind to one or more Fcγ receptors. See, e.g.,Shields et al., 2001, J. Biol. Chem. 276:6591-6604, and Canfield andMorrison, 1991, J. Exp. Med. 173:1483-1491. The Fc region includes, forexample and not for limitation, amino acid residues in the hinge regionand the C_(H)2 domain. Substitution of one or more amino acid residuesin the Fc region or portion of an IgG constant region withnon-conservative amino acids can be expected to alter, i.e., reduce theaffinity of the Fc region-FcγR interaction. Methods for introducingnon-conservative amino acid substitutions in an antibody or derivativethereof or other binding agent are well known in the art.

Alternatively or additionally, cysteine residue(s) may be introduced inor in proximity to the Fc region or portion of an IgG constant region,thereby allowing interchain disulfide bond formation in this region.Such interchain disulfide bond formation can be expected to cause sterichindrance, thereby reducing the affinity of the Fc region-FcγR bindinginteraction. The cysteine residue(s) introduced in or in proximity tothe Fc region of an IgG constant region may also serve as sites forconjugation to therapeutic agents (i.e., coupling cytotoxic drugs usingthiol specific reagents such as maleimide derivatives of drugs). Thepresence of a therapeutic agent can be expected to cause sterichindrance, thereby reducing the affinity of the Fc region-FcγR bindinginteraction. Methods for introducing cysteine residues in an antibody orderivative thereof or other binding agent are well known in the art.

Alternatively or additionally, one or more N-linked glycosylation sitesmay be introduced in or in proximity to the Fc region of an IgG constantregion, thereby allowing post-translational glycosylation in thisregion. Such N-linked glycosylation can be expected to cause sterichindrance, thereby reducing the affinity of the Fc region-FcγR bindinginteraction. Methods for introducing N-linked glycosylation sites in anantibody or derivative thereof or other binding agent are well known inthe art.

A systemic substitution of solvent-exposed amino acids of human IgG1 Fcregion has generated IgG derivatives with altered FcγR bindingaffinities (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Forexample, when compared to parental IgG1, a subset of these derivativesinvolving substitutions at Thr256/Ser298, Ser298/Glu333, Ser298/Lys334,or Ser298/Glu333/Lys334 to Ala demonstrate increases in both bindingaffinity toward FcγR and ADCC activity (Shields et al., 2001, J. Biol.Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49). Incontrast, when compared to parental IgG1, a subset of these derivativesinvolving substitutions at Glu233 to Pro/Leu234 to Val/Leu235 to Ala andGly 236 deletion, Pro238 to Ala, Asp265 to Ala, Asn297 to Ala, Ala 327to Gln, or Pro329 to Ala demonstrate decreases in binding affinities toall FcγR; the Asp265 to Ala substitution also resulted in decreased ADCCactivity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Aminoacids in the hinge region and the C_(H)2 domain have been shown tocontribute to high affinity of human IgG for FcγR (Canfield andMorrison, 1991, J. Exp. Med. 173:1483-1491). These amino acid positions,or amino acids in proximity thereto, involved in mediating the Fcregion-FcγR binding interaction are potential targets for replacement bynon-conservative amino acids and/or introduction of one or morecysteines, and/or introduction of one or more N-linked glycosylationsites.

The in vivo half-life of an antibody can also impact on its effectorfunctions. In some embodiments, it is desirable to increase thehalf-life of an antibody to modify its therapeutic activities. In someembodiments, it is desirable to decrease the half-life of an antibody tomodify its therapeutic activities. FcRn is a receptor that isstructurally similar to MHC Class I antigen that non-covalentlyassociates with β2-microglobulin. FcRn regulates the catabolism of IgGsand their transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The IgG-FcRn interaction takes place at pH 6.0 (pH of intracellularvesicles) but not at pH 7.4 (pH of blood); this interaction enables IgGsto be recycled back to the circulation (Ghetie and Ward, 2000, Ann. Rev.Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113).The region on human IgG₁ involved in FcRn binding has been mapped(Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alaninesubstitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312,Glu380, Glu382, or Asn434 of human IgG₁ enhance FcRn binding (Shields etal., 2001, J. Biol. Chem. 276:6591-604). IgG₁ molecules harboring thesesubstitutions are expected to have longer serum half-lives.Consequently, these modified IgG₁ molecules may be able to carry outtheir effector functions, and hence exert their therapeutic efficacies,over a longer period of time compared to unmodified IgG₁.

In an embodiment, the binding agents of the present invention havingimpaired binding to one or more FcγR retain, at least to some extent,the ability to bind FcRn. In an embodiment, the binding agents, whichhave impaired binding to one or more FcγR, retain the ability to bindFcRn. The ability of an antibody or derivative thereof or other bindingagent to bind to FcRn can be measured by techniques known in the art(e.g., Shields et al., 2001, J. Biol. Chem. 276:6591-604).

A CD19 binding agent modified with respect to effector function may, insome embodiments, have improved internalization capability and/orincreased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al. J. Exp Med. 176:1191-1195(1992) and Shopes, B. J. Immunol. 148:2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch 53:2560-2565 (1993). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

In certain embodiments, cysteine residue(s) may be introduced in the Fcregion in order to affect the binding interaction of the Fc region withthe FcγRIIIa receptor. In some embodiments, an amino acid substitutionof the native amino acid to a cysteine residue is introduced at aminoacid position 239, 265, 269 or 327, according to the Kabat numberingsystem. In some embodiments, an amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position239 or 269, according to the Kabat numbering system. In someembodiments, an amino acid substitution of the native amino acid to acysteine residue is introduced at amino acid position 239, according tothe Kabat numbering system. In some embodiments, an amino acidsubstitution of the native amino acid to a cysteine residue isintroduced at amino acid position 265, according to the Kabat numberingsystem. In some embodiments, an amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position269, according to the Kabat numbering system. In some embodiments, anamino acid substitution of the native amino acid to a cysteine residueis introduced at amino acid position 327, according to the Kabatnumbering system.

In other embodiments, an amino acid substitution of the native aminoacid to a cysteine residue is introduced at amino acid position 236 or238, according to the Kabat numbering system. In some embodiments, anamino acid substitution of the native amino acid to a cysteine residueis introduced at amino acid position 236, according to the Kabatnumbering system. In some embodiments, an amino acid substitution of thenative amino acid to a cysteine residue is introduced at amino acidposition 238, according to the Kabat numbering system.

In other embodiments, an amino acid substitution of the native aminoacid to a cysteine residue is introduced at amino acid position 234,235, 237, 267, 298, 299, 326, 330, or 332, according to the Kabatnumbering system. In other embodiments, an amino acid substitution ofthe native amino acid to a cysteine residue is introduced at amino acidposition 237, 298, 299, 326, 330, or 332, according to the Kabatnumbering system. In other embodiments, an amino acid substitution ofthe native amino acid to a cysteine residue is introduced at amino acidposition 298, 299, 326 or 330, according to the Kabat numbering system.In some embodiments, an amino acid substitution of the native amino acidto a cysteine residue is introduced at amino acid position 234,according to the Kabat numbering system. In some embodiments, an aminoacid substitution of the native amino acid to a cysteine residue isintroduced at amino acid position 235, according to the Kabat numberingsystem. In some embodiments, an amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position237, according to the Kabat numbering system. In some embodiments, anamino acid substitution of the native amino acid to a cysteine residueis introduced at amino acid position 267, according to the Kabatnumbering system. In some embodiments, an amino acid substitution of thenative amino acid to a cysteine residue is introduced at amino acidposition 298, according to the Kabat numbering system. In someembodiments, an amino acid substitution of the native amino acid to acysteine residue is introduced at amino acid position 299, according tothe Kabat numbering system. In some embodiments, an amino acidsubstitution of the native amino acid to a cysteine residue isintroduced at amino acid position 326, according to the Kabat numberingsystem. In some embodiments, an amino acid substitution of the nativeamino acid to a cysteine residue is introduced at amino acid position330, according to the Kabat numbering system. In some embodiments, anamino acid substitution of the native amino acid to a cysteine residueis introduced at amino acid position 332, according to the Kabatnumbering system.

In some embodiments, to further increase the serum half life of theantibody or derivative thereof or other binding agent, one may modifyany salvage receptor binding epitope in the antibody or derivativethereof or other binding agent as described in U.S. Pat. No. 5,739,277,for example. As used herein, the term “salvage receptor binding epitope”refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1,IgG2, IgG3, or IgG4) that is responsible for increasing the in vivoserum half-life of the IgG molecule. Alternatively, the serum half-lifeof the antibody or derivative thereof or other binding agent may beincreased by modifying the Fc region of an antibody (e.g., IgG constantdomain) with respect to binding to Fc gamma (Fcγ) receptors, asdescribed infra.

Antibodies may be glycosylated at conserved positions in their constantregions (see, e.g., Jefferis and Lund, 1997, Chem. Immunol. 65:111-128;Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide sidechains of the immunoglobulins can affect the protein's function (see,e.g., Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard,1990, Biochem. 29:4175-4180), and the intramolecular interaction betweenportions of the glycoprotein which can affect the conformation andpresented three-dimensional surface of the glycoprotein (see, e.g.,Jefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech.7:409-416). Oligosaccharides may also serve to target a givenglycoprotein to certain molecules based upon specific recognitionstructures. For example, it has been reported that in agalactosylatedIgG, the oligosaccharide moiety ‘flips’ out of the inter-C_(H)2 spaceand terminal N-acetylglucosamine residues become available to bindmannose binding protein (see, e.g., Malhotra et al., 1995, Nature Med.1:237-243). Removal by glycopeptidase of the oligosaccharides fromCAMPATH-1H (a recombinant humanized murine monoclonal IgG1 antibodywhich recognizes the CDw52 antigen of human lymphocytes) produced inChinese Hamster Ovary (CHO) cells resulted in a complete reduction incomplement mediated lysis (CMCL) (Boyd et al., 1996, Mol. Immunol.32:1311-1318), while selective removal of sialic acid residues usingneuraminidase resulted in no loss of DMCL. Glycosylation of antibodieshas also been reported to affect ADCC. In particular, CHO cells withtetracycline-regulated expression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing formation of bisecting GlcNAc, wasreported to have improved ADCC activity (see, e.g., Umana et al., 1999,Mature Biotech. 17:176-180).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Glycosylation derivatives of antibodies are derivatives in which theglycosylation pattern of an antibody is altered. Certain antibodies ofthe present invention have altered glycosylation patterns. By alteringis meant deleting one or more carbohydrate moieties found in theantibody, adding one or more carbohydrate moieties to the antibody,changing the composition of glycosylation (i.e., glycosylation pattern),the extent of glycosylation, or the like. In certain embodiments, theantibodies of the present invention have reduced core fucosylation.

Addition of glycosylation sites to the antibody can be convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the original antibody (for O-linked glycosylation sites).Similarly, removal of glycosylation sites can be accomplished by aminoacid alteration within the native glycosylation sites of the antibody.

The amino acid sequence is usually altered by altering the underlyingnucleic acid sequence. These methods include, but are not limited to,isolation from a natural source (in the case of naturally-occurringamino acid sequence derivatives) or preparation byoligonucleotide-mediated (or site-directed) mutagenesis, PCRmutagenesis, or cassette mutagenesis of an earlier prepared derivativeor a non-derivative version of the antibody.

The glycosylation (including glycosylation pattern) of antibodies mayalso be altered without altering the amino acid sequence or theunderlying nucleotide sequence. Glycosylation largely depends on thehost cell used to express the antibody. Since the cell type used forexpression of recombinant glycoproteins, e.g., antibodies, as potentialtherapeutics is rarely the native cell, significant variations in theglycosylation pattern of the antibodies can be expected. See, e.g., Hseet al., 1997, J. Biol. Chem. 272:9062-9070. In addition to the choice ofhost cells, factors which affect glycosylation during recombinantproduction of antibodies include growth mode, media formulation, culturedensity, oxygenation, pH, purification schemes, and the like. Variousmethods have been proposed to alter the glycosylation pattern achievedin a particular host organism, including introducing or overexpressingcertain enzymes involved in oligosaccharide production (see, e.g., U.S.Pat. Nos. 5,047,335; 5,510,261; and 5,278,299). Glycosylation, orcertain types of glycosylation, can be enzymatically removed from theglycoprotein, for example using endoglycosidase H (Endo H). In addition,the recombinant host cell can be genetically engineered, e.g., madedefective in processing certain types of polysaccharides. These andsimilar techniques are well known in the art.

The glycosylation structure of antibodies can be readily analyzed byconventional techniques of carbohydrate analysis, including lectinchromatography, NMR, mass spectrometry, HPLC, GPC, monosaccharidecompositional analysis, sequential enzymatic digestion, and HPAEC-PAD,which uses high pH anion exchange chromatography to separateoligosaccharides based on charge. Methods for releasing oligosaccharidesfor analytical purposes are also known, and include, without limitation,enzymatic treatment (commonly performed using peptide-N-glycosidaseF/endo-β-galactosidase), elimination using harsh alkaline environment torelease mainly O-linked structures, and chemical methods using anhydroushydrazine to release both N- and O-linked oligosaccharides.

The following table provides a summary of the regions of chimeric andhumanized BU12 to which each sequence identifier (SEQ ID NO.)corresponds.

Nucleotide or SEQ MOLECULE Amino Acid ID NO Leader Sequence Amino Acid 1 (Heavy Chain Region) Heavy Chain Variable Region Amino Acid  2(VH2-70/J_(H)4 germline)/Also referred to as Variant HA Heavy ChainConstant domain Amino Acid  3 (IgG₁) Variant HB Amino Acid  4 HeavyChain Variable Region (VH2-70/J_(H)4 germline) Variant HC Amino Acid  5Heavy Chain Variable Region (VH2-70/J_(H)4 germline) Variant HD AminoAcid  6 Heavy Chain Variable Region (VH2-70/J_(H)4 germline) Variant HEAmino Acid  7 Heavy Chain Variable Region (VH2-70/J_(H)4 germline) HeavyChain Variable Region Amino Acid  8 (Murine) Heavy Chain Variable RegionAmino Acid  9 (VH4-31/J_(H)4 germline)/also referred to as Variant HFVariant HG Amino Acid 10 Heavy Chain Variable Region (VH4-31/J_(H)4germline) Variant HH Amino Acid 11 Heavy Chain Variable Region(VH4-31/J_(H)4 germline) Variant HI Amino Acid 12 Heavy Chain VariableRegion (VH4-31/J_(H)4 germline) Variant HJ Amino Acid 13 Heavy ChainVariable Region (VH4-31/J_(H)4 germline) Variant HK Amino Acid 14 HeavyChain Variable Region (VH4-31/J_(H)4 germline) Variant HL Amino Acid 15Heavy Chain Variable Region (VH4-31/J_(H)4 germline) Leader SequenceAmino Acid 16 (Light Chain Region) Light Chain Variable Region AminoAcid 17 (VL-L6/J_(k)2 germline)/Also referred to as Variant LA LightChain Constant domain Amino Acid 18 (Kappa domain) Variant LB Amino Acid19 Light Chain Variable Region (VL-L6/J_(k)2 germline) Variant LC AminoAcid 20 Light Chain Variable Region (VL-L6/J_(k)2 germline) Variant LDAmino Acid 21 Light Chain Variable Region (VL-L6/J_(k)2 germline)Variant LE Amino Acid 22 Light Chain Variable Region (VL-L6/J_(k)2germline) Variant LF Amino Acid 23 Light Chain Variable Region(VL-L6/J_(k)2 germline) Variant LG Amino Acid 24 Light Chain VariableRegion (VL-L6/J_(k)2 germline) Light Chain Variable Region Amino Acid 25(Murine) Light Chain Variable Region Amino Acid 26 (VL-A10/J_(k)2germline)/Also referred to as Variant LH domain Variant LI Amino Acid 27Light Chain Variable Region (VL-A10/J_(k)2 germline) Consensus sequencefor Heavy Amino Acid 28 Chain Variable Region (VH2-70/J_(H)4 germline)Consensus sequence for Heavy Amino Acid 29 Chain Variable Region(VH4-31/J_(H)4 germline) Consensus sequence for Light Amino Acid 30Chain Variable Region (VL-L6/J_(k)2 germline) Consensus sequence forLight Amino Acid 31 Chain Variable Region (VL-A10/J_(k)2 germline)Consensus sequence for Heavy Amino Acid 32 Chain Variable Region(VH2-70/J_(H)1-6 germline) Consensus sequence for Heavy Amino Acid 33Chain Variable Region (VH4-31/J_(H)1-6 germline) Consensus sequence forLight Amino Acid 34 Chain Variable Region (VL-L6/J_(k)1-5 germline)Consensus sequence for Light Amino Acid 35 Chain Variable Region(VL-A10/J_(k)1-5 germline) Heavy Chain Constant Domain Amino Acid 36(IgG₂) Heavy Chain Constant Domain Amino Acid 37 (IgG₃) Heavy ChainConstant Domain Amino Acid 38 (IgG₄) Heavy Chain Constant Domain AminoAcid 39 Variant (IgG₁V₁) Leader Sequence Nucleotide 40 (Heavy ChainRegion) Heavy Chain Variable Region Nucleotide 41 (VH4-31/J_(H)4germline)/also referred to as Variant HF Heavy Chain Constant domainNucleotide 42 (IgG₁) Leader Sequence Nucleotide 43 (Light Chain Region)Variant LG Nucleotide 44 Light Chain Variable Region (VL-L6/J_(k)2germline) Light Chain Constant domain Nucleotide 45 (Kappa domain) HeavyChain CDR1 Amino Acid 46 Heavy Chain CDR2 Amino Acid 47 Heavy Chain CDR3Amino Acid 48 Light Chain CDR1 Amino Acid 49 Light Chain CDR2 Amino Acid50 Light Chain CDR3 Amino Acid 51

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:2. The aminoacid sequence can be, for example, the amino acid sequence of SEQ IDNO:2 having any number of substitutions provided that the CD19 bindingagent retains functional activity (i.e., CD19 binding activity) and thatthe sequence retains substantial or complete identity to the amino acidsequence set forth in SEQ ID NO:2. Exemplary heavy chain variableregions comprise an amino acid sequence that is identical to the aminoacid sequence set forth in SEQ ID NO:2 optionally having at least oneamino acid substitution, preferably 0, 1 or 2 amino acid substitutions,at positions 75, 79, 81, 82, 82A, 82B, 82C or 89 of the amino acidsequence set forth in SEQ ID NO:2, according to the Kabat numberingsystems. Exemplary sequences include, for example, the amino acidsequences set forth in SEQ ID NOs:2, 4, 5, 6, and 7. In one aspect, theCD19 binding agent that comprises a heavy chain variable regioncomprising an amino acid sequence that is identical or substantiallyidentical (i.e., having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity) to the amino acid sequence set forth in SEQID NO:2 comprises the CDR regions of the antibody mBU12, i.e., SEQ IDNO:46, 47, and 48.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:9. The aminoacid sequence can be, for example, the amino acid sequence of SEQ IDNO:9 having any number of substitutions provided that the CD19 bindingagent retains functional activity and that the sequence retainssubstantial or complete identity to the amino acid sequence set forth inSEQ ID NO:9. Exemplary heavy chain variable regions comprise an aminoacid sequence that is identical to the amino acid sequence set forth inSEQ ID NO:9 optionally having at least one amino acid substitution,preferably 0, 1 or 2 amino acid substitutions, at positions 24, 27, 29,71, 75, 78, 79, or 89 of the amino acid sequence set forth in SEQ IDNO:9, according to the Kabat numbering systems. Exemplary sequencesinclude, for example, the amino acid sequences set forth in SEQ IDNOs:9, 10, 11, 12, 13, 14, and 15. In one aspect, the CD19 binding agentthat comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:9 comprisesthe CDR regions of antibody mBU12, i.e., SEQ ID NO:46, 47, and 48.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a light chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:17. Theamino acid sequence can be, for example, the amino acid sequence of SEQID NO:17 having any number of substitutions provided that the CD19binding agent retains functional activity and that the sequence retainssubstantial or complete identity to the amino acid sequence set forth inSEQ ID NO:17. Exemplary light chain variable regions comprise an aminoacid sequence that is identical to the amino acid sequence set forth inSEQ ID NO:17 optionally having at least one amino acid substitution,preferably 0, 1 or 2 amino acid substitutions, at positions 2, 40, 41,42, 69, 70, 71, 72 and 83 of the amino acid sequence set forth in SEQ IDNO:17, according to the Kabat numbering systems. Exemplary sequencesinclude, for example, the amino acid sequences set forth in SEQ IDNOs:17, 19, 20, 21, 22, 23, and 24. In one aspect, the CD19 bindingagent that comprises a light chain variable region comprising an aminoacid sequence that is identical or substantially identical (i.e., havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:17 comprisesthe CDR regions of the antibody mBU12, i.e., SEQ ID NO:49 50, and 51.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a light chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:26. Theamino acid sequence can be, for example, the amino acid sequence of SEQID NO:26 having any number of substitutions provided that the CD19binding agent retains functional activity and that the sequence retainssubstantial or complete identity to the amino acid sequence set forth inSEQ ID NO:26. Exemplary light chain variable regions comprise an aminoacid sequence that is identical to the amino acid sequence set forth inSEQ ID NO:26 optionally having at least one amino acid substitution atpositions 2 and 71 of the amino acid sequence set forth in SEQ ID NO:26,according to the Kabat numbering systems. Exemplary sequences include,for example, the amino acid sequences set forth in SEQ ID NOs:26 and 27.In one aspect, the CD19 binding agent that comprises a light chainvariable region comprising an amino acid sequence that is identical orsubstantially identical (i.e., having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequenceset forth in SEQ ID NO:26 comprises the CDR regions of the antibodymBU12, i.e., SEQ ID NO:49, 50, and 51.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to SEQ ID NO:2 as provided above, and further comprises alight chain variable region comprising an amino acid sequence that isidentical or substantially identical (i.e., having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ IDNO:17, as provided above. In any of these embodiments, the heavy chainvariable region can be joined to a constant region as set forth, forexample, in SEQ ID NOs:3, or 36-39 and the light chain variable regioncan be joined to a constant region as set forth, for example, in SEQ IDNO:18. In certain embodiments, the heavy chain variable region willfurther comprise the amino acid sequence set forth in SEQ ID NO:1 andthe light chain variable region will further comprise the amino acidsequence set forth in SEQ ID NO:16.

Accordingly, in certain embodiments, the CD19 binding agent willcomprise a heavy chain comprising the amino acid sequence set forth inSEQ ID NO:2 and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:4 and a lightchain comprising the amino acid sequence set forth in SEQ ID NO: 17, 19,20, 21, 22, 23 or 24; a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO:5 and a light chain comprising the amino acidsequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a heavychain comprising the amino acid sequence set forth in SEQ ID NO:6 and alight chain comprising the amino acid sequence set forth in SEQ ID NO:17, 19, 20, 21, 22, 23 or 24; or a heavy chain comprising the amino acidsequence set forth in SEQ ID NO:7 and a light chain comprising the aminoacid sequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24. Inany of these embodiments, the heavy chain variable region can be joinedto a constant region as set forth, for example, in SEQ ID NOs:3, or36-39 and the light chain variable region can be joined to a constantregion as set forth, for example, in SEQ ID NO:18. In certainembodiments, the heavy chain variable region will further comprise theamino acid sequence set forth in SEQ ID NO:1 and the light chainvariable region will further comprise the amino acid sequence set forthin SEQ ID NO:16.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:2 asprovided above, and further comprises a light chain variable regioncomprising an amino acid sequence that is identical or substantiallyidentical (i.e., having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity) to the amino acid sequence set forth in SEQID NO:26, as provided above. In any of these embodiments, the heavychain variable region can be joined to a constant region as set forth,for example, in SEQ ID NOs:3, or 36-39 and the light chain variableregion can be joined to a constant region as set forth, for example, inSEQ ID NO:18. In certain embodiments, the heavy chain variable regionwill further comprise the amino acid sequence set forth in SEQ ID NO:1and the light chain variable region will further comprise the amino acidsequence set forth in SEQ ID NO:16.

Accordingly, in certain embodiments, the CD19 binding agent willcomprise a heavy chain comprising the amino acid sequence set forth inSEQ ID NO:2 and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 26 or 27; a heavy chain comprising the amino acidsequence set forth in SEQ ID NO:4 and a light chain comprising the aminoacid sequence set forth in SEQ ID NO: 26 or 27; a heavy chain comprisingthe amino acid sequence set forth in SEQ ID NO:5 and a light chaincomprising the amino acid sequence set forth in SEQ ID NO: 26 or 27; aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:6and a light chain comprising the amino acid sequence set forth in SEQ IDNO: 26 or 27; or a heavy chain comprising the amino acid sequence setforth in SEQ ID NO:7 and a light chain comprising the amino acidsequence set forth in SEQ ID NO: 26 or 27. In any of these embodiments,the heavy chain variable region can be joined to a constant region asset forth, for example, in SEQ ID NOs:3, or 36-39 and the light chainvariable region can be joined to a constant region as set forth, forexample, in SEQ ID NO:18. In certain embodiments, the heavy chainvariable region will further comprise the amino acid sequence set forthin SEQ ID NO:1 and the light chain variable region will further comprisethe amino acid sequence set forth in SEQ ID NO:16.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:9 asprovided above, and further comprises a light chain variable regioncomprising an amino acid sequence that is identical or substantiallyidentical (i.e., having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity) to SEQ ID NO:17, as provided above. In anyof these embodiments, the heavy chain variable region can be joined to aconstant region as set forth, for example, in SEQ ID NOs:3, or 36-39 andthe light chain variable region can be joined to a constant region asset forth, for example, in SEQ ID NO:18. In certain embodiments, theheavy chain variable region will further comprise the amino acidsequence set forth in SEQ ID NO:1 and the light chain variable regionwill further comprise the amino acid sequence set forth in SEQ ID NO:16.

Accordingly, in certain embodiments, the CD19 binding agent willcomprise a heavy chain comprising the amino acid sequence set forth inSEQ ID NO:9 and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:10 and a lightchain comprising the amino acid sequence set forth in SEQ ID NO: 17, 19,20, 21, 22, 23 or 24; a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO:11 and a light chain comprising the amino acidsequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a heavychain comprising the amino acid sequence set forth in SEQ ID NO:12 and alight chain comprising the amino acid sequence set forth in SEQ ID NO:17, 19, 20, 21, 22, 23 or 24; a heavy chain comprising the amino acidsequence set forth in SEQ ID NO:13 and a light chain comprising theamino acid sequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24a heavy chain comprising the amino acid sequence set forth in SEQ IDNO:14 and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 17, 19, 20, 21, 22, 23 or 24 or a heavy chain comprising theamino acid sequence set forth in SEQ ID NO:15 and a light chaincomprising the amino acid sequence set forth in SEQ ID NO: 17, 19, 20,21, 22, 23 or 24. In any of these embodiments, the heavy chain variableregion can be joined to a constant region as set forth, for example, inSEQ ID NOs:3, or 36-39 and the light chain variable region can be joinedto a constant region as set forth, for example, in SEQ ID NO:18. Incertain embodiments, the heavy chain variable region will furthercomprise the amino acid sequence set forth in SEQ ID NO:1 and the lightchain variable region will further comprise the amino acid sequence setforth in SEQ ID NO:16.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:9 asprovided above, and further comprises a light chain variable regioncomprising an amino acid sequence that is identical or substantiallyidentical (i.e., having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identity) to the amino acid sequence set forth in SEQID NO:26, as provided above. In any of these embodiments, the heavychain variable region can be joined to a constant region as set forth,for example, in SEQ ID NOs:3, or 36-39 and the light chain variableregion can be joined to a constant region as set forth, for example, inSEQ ID NO:18. In certain embodiments, the heavy chain variable regionwill further comprise the amino acid sequence set forth in SEQ ID NO:1and the light chain variable region will further comprise the amino acidsequence set forth in SEQ ID NO:16.

Accordingly, in certain embodiments, the CD19 binding agent willcomprise a heavy chain comprising the amino acid sequence set forth inSEQ ID NO:9 and a light chain comprising the amino acid sequence setforth in SEQ ID NO: 26 or 27; a heavy chain comprising the amino acidsequence set forth in SEQ ID NO:10 and a light chain comprising theamino acid sequence set forth in SEQ ID NO: 26 or 27; a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO:11 and a lightchain comprising the amino acid sequence set forth in SEQ ID NO: 26 or27; a heavy chain comprising the amino acid sequence set forth in SEQ IDNO:12 and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 26 or 27; a heavy chain comprising the amino acid sequenceset forth in SEQ ID NO:13 and a light chain comprising the amino acidsequence set forth in SEQ ID NO: 26 or 27; heavy chain comprising theamino acid sequence set forth in SEQ ID NO:14 and a light chaincomprising the amino acid sequence set forth in SEQ ID NO: 26 or 27; ora heavy chain comprising the amino acid sequence set forth in SEQ IDNO:15 and a light chain comprising the amino acid sequence set forth inSEQ ID NO: 26 or 27. In any of these embodiments, the heavy chainvariable region can be joined to a constant region as set forth, forexample, in SEQ ID NOs:3, or 36-39 and the light chain variable regioncan be joined to a constant region as set forth, for example, in SEQ IDNO:18. In certain embodiments, the heavy chain variable region willfurther comprise the amino acid sequence set forth in SEQ ID NO:1 andthe light chain variable region will further comprise the amino acidsequence set forth in SEQ ID NO:16.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:28 or SEQ IDNO:32. The amino acid sequence can be, for example, the amino acidsequence of SEQ ID NO:28 or SEQ ID NO:32 having any number ofsubstitutions provided that the CD19 binding agent retains functionalactivity (i.e., CD19 binding activity) and that the sequence retainssubstantial or complete identity to the amino acid sequence set forth inSEQ ID NO:28 or SEQ ID NO:32, respectively.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:29 or SEQ IDNO:33. The amino acid sequence can be, for example, the amino acidsequence of SEQ ID NO:29 or SEQ ID NO:33 having any number ofsubstitutions provided that the CD19 binding agent retains functionalactivity and that the sequence retains substantial or complete identityto the amino acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO:33,respectively.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a light chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:30 or SEQ IDNO:34. The amino acid sequence can be, for example, the amino acidsequence of SEQ ID NO: 30 or SEQ ID NO:34 having any number ofsubstitutions provided that the CD19 binding agent retains functionalactivity and that the sequence retains substantial or complete identityto the amino acid sequence set forth in SEQ ID NO: 30 or SEQ ID NO:34,respectively.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a light chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:31 or SEQ IDNO:35. The amino acid sequence can be, for example, the amino acidsequence of SEQ ID NO:31 or SEQ ID NO:35 having any number ofsubstitutions provided that the CD19 binding agent retains functionalactivity and that the sequence retains substantial or complete identityto the amino acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO:35,respectively.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to SEQ ID NO:28 or SEQ ID NO:32 as provided above, and furthercomprises a light chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to SEQ ID NO:30 or SEQ ID NO:34, as provided above.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:28 or SEQ IDNO:32 as provided above, and further comprises a light chain variableregion comprising an amino acid sequence that is identical orsubstantially identical (i.e., having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequenceset forth in SEQ ID NO:31 or SEQ ID NO:35, as provided above.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:29 or SEQ IDNO:33 as provided above, and further comprises a light chain variableregion comprising an amino acid sequence that is identical orsubstantially identical (i.e., having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:30 or SEQ IDNO:34 as provided above.

A humanized antibody comprising a heavy chain variable region of SEQ IDNO:9 and a light chain variable region of SEQ ID NO:17 has all sixintact CDRs from the mouse BU12 antibody and entirely human variableregion framework amino acids. In contrast to many other humanizedantibodies, such an antibody has useful binding affinity to its antigeneven without any further substitutions. However, such an antibody alsoprovides a starting point for making variants. Some such variantscomprise a heavy chain variable region having at least 90% sequenceidentity (spanning its entire length) to SEQ ID NO:9 and a light chainvariable region having at least 90% sequence identity to SEQ ID NO:17.Some variants have no more than 5, 4, 3, 2 or 1 amino acids that differfrom SEQ ID NO:9 in the heavy chain variable region and no more than 5,4, 3, 2, or 1 amino acids that differ from SEQ ID NO:17 in the lightchain variable region.

Some variants differ from the above antibody by substitution of one ormore amino acids in the variable region framework relative to SEQ IDNO:9 or SEQ ID NO:17. The substitution can be with an amino acidoccupying the corresponding position (unless otherwise indicatedpositions are by Kabat numbering) in the heavy or light chain variableregion respectively of the BU12 antibody (sometimes referred to as adonor antibody). Substitutions with donor amino acids often increase theaffinity of the resulting humanized antibody for its antigen byconferring a framework conformation in the humanized antibody moreclosely resembling that in the donor mouse antibody. A donorsubstitution at position L83 is particularly advantageous in increasingaffinity as shown in FIG. 2 (compare light chain G with the substitutionand H without the substitution). Other positions differing between SEQID NO:2 and the BU12 heavy chain in which substitutions can be performedinclude H3, H24, H27, H29, H41, H71, H75, H789, H79, H83 and H89 inwhich the positions are occupied by Q, V, G, I, P, V, K, F, P, T and Vrespectively. Light chain variable region framework positions differingbetween SEQ ID NO:17 and the BU12 light chain include positions L2, L40,L41, L42, L69, L70, L71, L72 and L83 occupied by I, P, G, Q, T, D, F, Tand F respectively in the mouse BU12 light chain. The effect of many ofthese substitutions on antibody affinity is also shown in FIG. 2. It canbe seen that some of these substitutions or combinations ofsubstitutions increase affinity. Some preferred heavy chain substitutioninclude one or more of H71, H75, H78 and H79 occupied by V, K, F and Prespectively. Some preferred light chain substitutions include one ormore of positions L2, L69, L71, L72 and L83 is occupied by I, T, F, Tand F respectively. None of the tested substitutions or combinations ofsubstitutions caused an unacceptable loss of affinity.

How many donor substitutions to include reflects a balance of competingconsiderations. In general, substitutions which significantly increaseaffinity are desirable. However, minimizing the total number of variableregion framework substitutions is also advantageous in reducingpotential immunogenicity. A humanized antibody having no substitutionsin the heavy chain variable region framework and a L83 donorsubstitution of the light chain variable region framework represents apreferred balance between maximizing affinity and minimizingimmunogenicity. Many other permutations are possible.

As well as or instead of donor substitutions, a variable regionframework amino acid can be substituted with the amino acid occupyingthe corresponding position in another human antibody sequence or aconsensus of human antibody sequences (see, .e.g., Queen, U.S. Pat. No.7,022,500). The rationale for such a substitution is often to substitutea relatively rare amino acid in human immunoglobulin sequences with amore common amino acid for that position with a view to reducingimmunogenicity. In humanized antibodies in which the variable regionframeworks are provided by germline sequences, such substitutions arepossible but generally not necessary because germline sequences lackrare amino acids that may be introduced by somatic mutation.

Variable region framework amino acids can also be substituted with aminoacids that are neither donor amino acids or consensus amino acids. Suchsubstitutions are preferably conservative amino acid substitutions.Although many substitutions have little effect on affinity, they mayincrease immunogenicity and thus in general are not preferred.

Substitution of one or more CDR residues or omission of one or more CDRsis also possible. Numerous antibodies have been described in thescientific literature in which one or two CDRs can be dispensed with forbinding. Padlan et al., FASEB Journal 9: 133-139 (1995) analyzed thecontact regions between antibodies and their antigens, based onpublished crystal structures, and concluded that only about one fifth toone third of CDR residues actually contact the antigen. Padlan et al.termed these residues SDR (for specificity determining residues). Padlanalso found many antibodies in which one or two CDRs had no amino acidsin contact with an antigen. Likewise, Vajdos et al (Journal of MolecularBiology, vol. 320, pp. 415-428 (2002) reported that CDR1 of the lightchain of an antibody against ErbB2 was not involved in binding. Suchteaching has been applied to antibody humanization by, for example,Iwahashi et al., Mol. Immunol. 36:1079-1091, (1999), who showed thatthey could graft only L1 and L3, or L2 and L3, of the CR49 murineantibody onto a human framework and retain high affinity interactionwith the antigen. Similarly, Tamura et al, Journal of Immunology, 2000,164:1432-1441 (2000) reported that light chain CDRs 1 and 2 could bedispensed with entirely in a humanized anti-carcinoma antibody, as couldseveral residues in the remaining CDRs. The substitution of certainregions within CDRs is based on the same principle as omittingdispensable CDRs, namely that only a small subset of CDR residues, theSDR's, actually contact antigen.

CDR residues not contacting antigen can be identified based on previousstudies (for example residues H60-H65 in CDRH2 are often not required),from regions of Kabat CDRs lying outside Chothia CDRs, by molecularmodeling and/or empirically. If a CDR or residue(s) thereof is omitted,it is usually substituted with an amino acid occupying the correspondingposition in human acceptor sequence supplying the variable regionframework sequences (in this example, VH4-31/JH4 for the heavy chain andVL-L6 JK2 for the light chain). The number of such substitutions toinclude reflects a balance of competing considerations Suchsubstitutions are potentially advantageous in decreasing the number ofmouse amino acids in a humanized antibody and consequently decreasingpotential immunogenicity. However, substitutions can also cause changesof affinity, and significant reductions in affinity are preferablyavoided. Positions for substitution within CDRs and amino acids tosubstitute can also be selected empirically. Empirical substitutions canbe conservative or non-conservative substitutions. However, in generalempirical substitutions do not have the advantage of mouse to humansubstitutions in reducing immunogenicity. Empirical substitutions canincrease or decrease affinity of the resulting humanized antibody.

In general humanized antibodies with satisfactory binding affinity toCD19 and lack of substantial immunogenicity can be obtained byindividual screening of a view variants made according to the aboveprinciples and/or in accordance with the present examples. However, verylarge numbers of variants can be simultaneously screened using a displayselection method such as phage display (see (Dower et al., WO91/17271;McCafferty et al., WO92/001047; and Winter, WO92/20791). The sameconsiderations apply mutatis mutandis in designing variants of otherhumanized antibodies or antibody chains disclosed herein. For example,SEQ ID NO:2 provides an alternative starting point to SEQ ID NO:9 fordesign of heavy chain variants. SEQ ID NO:2 comprises the three heavychain CDRs of the BU12 antibody with a fully human variable regionframework sequence of the VH2-70 and JH4 genes. SEQ ID NO:26 provides analternative starting point to SEQ ID NO:17 for design of light chainvariants. SEQ ID NO:26 comprises the three light chain CDRs of the BU12antibody in a fully human framework sequence of the A10 and JK2 genes.Specific heavy chain variable region framework positions for potentialsubstitution and the amino acids to substitute into such positions areindicated in the table “non-homologous FR residues BU12 v.s VH431) inExample. Specific light chain variable region framework positions forpotential substitution in SEQ ID NO:26 are indicated in the table“Nono-homologous FR residues BU12 VL vs. L6 and A10” in Example 2.

The present invention encompasses embodiments wherein the CD19 bindingagent comprises a heavy chain variable region comprising an amino acidsequence that is identical or substantially identical (i.e., having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity) to the amino acid sequence set forth in SEQ ID NO:29 or SEQ IDNO:33 as provided above, and further comprises a light chain variableregion comprising an amino acid sequence that is identical orsubstantially identical (i.e., having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid sequenceset forth in SEQ ID NO:31 or SEQ ID NO:35, as provided above.

The present invention encompasses embodiments wherein the heavy chainvariable region further comprises a leader sequence. One such leadersequence is set forth in SEQ ID NO:1.

The present invention encompasses embodiments wherein the light chainvariable region further comprises a leader sequence. One such leadersequence is set forth in SEQ ID NO:16.

The CD19 binding agent can optionally include an antibody effectorregion. The effector domain(s) can be, for example, an Fc region such asa hinge-C_(H)2-C_(H)3 region of an immunoglobulin, or a portion orfragment of an effector region preferably having effector function.Antigen-binding antibody fragments, including single-chain antibodies,can comprise, for example, the variable region(s) in combination withthe entirety or a portion of an effector region (e.g., a C_(H)2 and/orC_(H)3 domain alone or in combination with a C_(H)1, hinge and/or C_(L)domain). Also, antigen-binding fragments can comprise any combination ofeffector regions. In some embodiments, the anti-CD19 antibody can be asingle chain antibody comprising a CD19-binding variable region joinedto hinge-C_(H)2-C_(H)3 domains.

The effector region of the anti-CD19 antibody can be from any suitableimmunoglobulin isotype. A CD19 binding agent can be expressed as arecombinant fusion protein comprising of the appropriate constantdomains to yield the desired effector function(s).

The present invention encompasses embodiments wherein the heavy chainvariable region of a CD19 binding agent is joined to a constant region,such an IgG, i.e., IgG1 constant region or IgG2 constant region, oraltered constant region, e.g., IgG1V1. Exemplary constant region domainsare provided as SEQ ID NO:3 and 36-39.

The present invention also encompasses embodiments wherein the lightchain variable region of a CD19 binding agent is joined to a constantregion, such as a kappa constant region. An exemplary constant regiondomain is provided as SEQ ID NO:18.

The present invention encompasses embodiments wherein the light chainvariable region of a CD19 binding agent is joined to a constant region,such as a kappa constant region. An exemplary constant region domain isprovided as SEQ ID NO:18 and the heavy chain variable region of a CD19binding agent is joined to a constant region, such an IgG, i.e., IgG1constant region or IgG2 constant region, or altered constant region,e.g., IgG1V1. Exemplary constant region domains are provided as SEQ IDNO: 3 and 36-39.

In some embodiments, a CD19 binding agent can be a CD19 binding agent,comprising a human or non-human Fc region or portion thereof. Forexample, the antibody can include an Fc region or portion thereof ofnon-human origin, e.g., rodent (e.g., mouse or rat), donkey, sheep,rabbit, goat, guinea pig, camelid, horse, chicken or monkey (e.g.,macaque, rhesus, cynomolgous or the like) linked to humanized heavyand/or light chain variable regions.

A CD19 binding agent, such as an antibody, can be monospecific,bispecific, trispecific, or of greater multispecificity. Multispecificantibodies may be specific for different epitopes of CD19 and/or may bespecific for both CD19 as well as for a heterologous protein. (See,e.g., PCT Publications WO 93/17715, WO 92/08802, WO 91/00360, and WO92/05793; Tutt et al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; and U.S. Pat. No. 5,601,819;Kostelny et al., 1992, J. Immunol. 148:1547-1553.) Multispecificantibodies, including bispecific and trispecific antibodies, useful forpracticing the methods described herein are antibodies thatimmunospecifically bind to both CD19 and a second cell surface receptoror receptor complex, i.e., one that mediates ADCC, ADCP, and/or CDC.

CD19 binding agents may also be described or specified in terms of theirbinding affinity to CD19. Typical binding affinities include those witha dissociation constant or Kd less than 5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁻⁷M, 5×10⁻⁸M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M,10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×⁻¹³ M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M,5×10⁻¹⁵M, or 10⁻¹⁵ M.

The present invention encompasses nucleic acids encoding a CD19 bindingagent. The CD19 binding agent can be, for example, a fully humanizedantibody or a humanized antigen-binding fragment. In some embodiments,the nucleic acid encodes a polypeptide chain having the amino acidsequence or having substantial identity to the amino acid sequences setforth in SEQ ID NOs: 2, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19,20, 21, 22, 23, 24, 26 or 27. In certain embodiments, a nucleic acid ofthe present invention will comprise the nucleotide sequence set forth inSEQ ID NOS. 40, 41, 42, 43, 44, or 45. In certain embodiments, thenucleic acid will encode a heavy chain variable region of an antibodyand will comprise one or more of the sequences set forth in SEQ ID NOS.40, 41, and 42. In certain embodiments, the nucleic acid will encode alight chain variable region of an antibody and will comprise one or moreof the sequences set forth in SEQ ID NOS. 43, 44, and 45.

Also included in some embodiments are nucleic acids encoding a CD19binding agent that hybridize under low, moderate, and high stringencyconditions, as defined herein, to all or a portion of a nucleotidesequence encoding a CD19 binding agent disclosed herein, or by itscomplement.

The present invention encompasses embodiments wherein the CD19 bindingagent is, for example, a humanized full length antibody, humanizedantibody fragment, or a derivative thereof.

CD19 binding agents can be generated by methods known in the art. Forexample, monoclonal antibodies can be prepared using a wide variety oftechniques including, e.g., the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. Hybridoma techniques aregenerally discussed in, for example, Harlow et al., Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed., 1988;Harlow and Lane, Using Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1999); and Hammerling et al., In MonoclonalAntibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y., 1981).Examples of phage display methods that can be used to make anti-CD19antibodies include, e.g., those disclosed in Hoogenboom and Winter,1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581;Quan and Carter, 2002, The rise of monoclonal antibodies as therapeuticsin Anti-IgE and Allergic Disease, Jardieu and Fick Jr., eds., MarcelDekker, New York, N.Y., Chapter 20, pp. 427-469; Brinkman et al., 1995,J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958;Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances inImmunology 57:191-280; PCT Application No. PCT/GB91/01134; PCTPublications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO93/11236, WO 95/15982, WO 95/20401, and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108 (the disclosures of which are incorporated by referenceherein).

As discussed herein, the CD19 binding agents can include the amino acidsequence of a humanized heavy and/or light chain variable region.Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (see, e.g., EP 0 239 400; PCTpublication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and5,585,089), veneering or resurfacing (see, e.g., EP 0 592 106; EP 0 519596; Padlan, Molecular Immunology, 1991, 28(4/5):489-498; Studnicka etal., 1994, Protein Engineering 7(6):805-814; Roguska et al., 1994, Proc.Natl. Acad. Sci. USA 91:969-973), and chain shuffling (see, e.g., U.S.Pat. No. 5,565,332) (all of these references are incorporated byreference herein). Humanized antibodies and fragments thereof can beproduced by recombinant DNA techniques known in the art, for exampleusing methods described in International Publication No. WO 87/02671;European Patent Publication No. 0 184 187; European Patent PublicationNo. 0 171 496; European Patent Publication No. 0 173 494; InternationalPublication No. WO 86/01533; U.S. Pat. No. 4,816,567; European PatentPublication No. 0 012 023; Berter et al., 1988, Science 240:1041-43; Liuet al., 1987, Proc. Natl. Acad. Sci. USA 84:3439-43; Liu et al., 1987,J. Immunol. 139:3521-26; Sun et al., 1987, Proc. Natl. Acad. Sci. USA84:214-18; Nishimura et al., 1987, Cancer. Res. 47:999-1005; Wood etal., 1985, Nature 314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst.80:1553-59; Morrison, 1985, Science 229:1202-07; Oi et al., 1986,BioTechniques 4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature321:552-25; Verhoeyan et al., 1988, Science 239:1534; and Beidler etal., 1988, J. Immunol. 141:4053-60; each of which is incorporated hereinby reference in its entirety.

Examples of techniques that can be used to produce single-chainantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88; Shu etal., 1993, Proc. Natl. Acad. Sci. USA 90:7995-7999; and Skerra et al.,1988, Science 240:1038-1040.

Methods for making bispecific antibodies are known in the art.Traditional production of full-length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (see, e.g., Milsteinet al., 1983, Nature 305:537-39). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of different antibody molecules, of whichone has the correct bispecific structure. Similar procedures aredisclosed in International Publication No. WO 93/08829, and inTraunecker et al., 1991, EMBO J. 10:3655-59.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion typicallyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, C_(H)2, and C_(H)3 regions. In someembodiments, the fusion includes a first heavy-chain constant region(C_(H)1) containing the site necessary for light chain binding, presentin at least one of the fusions. Nucleic acids with sequences encodingthe immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In an example of this approach, the bispecific antibodies have a hybridimmunoglobulin heavy chain with a first binding specificity in one arm,and a hybrid immunoglobulin heavy chain-light chain pair (providing asecond binding specificity) in the other arm. This asymmetric structurefacilitates the separation of the desired bispecific compound fromunwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation (see, e.g., InternationalPublication No. WO 94/04690, which is incorporated herein by referencein its entirety).

For further discussion of bispecific antibodies see, for example, Sureshet al., 1986, Methods in Enzymology 121:210; Rodrigues et al., 1993, J.Immunology 151:6954-61; Carter et al., 1992, Bio/Technology 10:163-67;Carter et al., 1995, J. Hematotherapy 4:463-70; Merchant et al., 1998,Nature Biotechnology 16:677-81. Using such techniques, bispecificantibodies can be prepared for use in the treatment or prevention ofdisease as defined herein.

Bifunctional antibodies are also described in European PatentPublication No. 0 105 360. As disclosed in this reference, hybrid orbifunctional antibodies can be derived biologically, i.e., by cellfusion techniques, or chemically, especially with cross-linking agentsor disulfide-bridge forming reagents, and may comprise whole antibodiesor fragments thereof. Methods for obtaining such hybrid antibodies aredisclosed for example in International Publication WO 83/03679 andEuropean Patent Publication No. 0 217 577, both of which areincorporated herein by reference.

A CD19 binding agent can be a derivative of an anti-CD19 antibody. Incertain embodiments, an anti-CD19 antibody derivative comprises ananti-CD19 antibody (e.g., an intact antibody, an antigen-bindingfragment or conservatively substituted polypeptide) and at least onepolypeptide region or other moiety heterologous to the anti-CD19antibody. For example, an anti-CD19 antibody can be modified, e.g., bythe covalent attachment of any type of molecule, such that the covalentattachment does not prevent the antibody derivative from specificallybinding to CD19 via the antigen-binding region or region derivedtherefrom, or, if desired, the effector region or portion thereof fromspecifically binding Fc receptor. Typical modifications include, e.g.,glycosylation, deglycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, and the like. Any of numerous chemical modifications may becarried out by known techniques, including, but not limited to specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc.

In some embodiments, the antibody derivative is a multimer, such as, forexample, a dimer, comprising one or more monomers, where each monomerincludes (i) an antigen-binding region of an anti-CD19 antibody, or apolypeptide region derived therefrom (such as, for example, byconservative substitution of one or more amino acids), and (ii) amultimerizing (e.g., dimerizing) polypeptide region, such that theantibody derivative forms multimers (e.g., homodimers) that specificallybind to CD19. In typical embodiments, an antigen binding region of ananti-CD19 antibody, or a polypeptide region derived therefrom, isrecombinantly or chemically fused with a heterologous protein, whereinthe heterologous protein comprises a dimerization or multimerizationdomain. Prior to administration of the antibody derivative to a subjectfor the purpose of treating or preventing CD19-expressing cancers, thederivative is subjected to conditions that allow formation of ahomodimer or heterodimer. A heterodimer, as used herein, may compriseidentical dimerization domains but different CD19 antigen-bindingregions, identical CD19 antigen-binding regions but differentdimerization domains, or different CD19 antigen-binding regions anddimerization domains.

Typical dimerization domains are those that originate from transcriptionfactors. In one embodiment, the dimerization domain is that of a basicregion leucine zipper (“bZIP”) (see, e.g., Vinson et al., 1989, Science246:911-916). Useful leucine zipper domains include, for example, thoseof the yeast transcription factor GCN4, the mammalian transcriptionfactor CCAAT/enhancer-binding protein C/EBP, and the nuclear transformin oncogene products, Fos and Jun. (See, e.g., Landschultz et al., 1988,Science 240:1759-64; Baxevanis and Vinson, 1993, Curr. Op. Gen. Devel.3:278-285; O'Shea et al., 1989, Science 243:538-542.) In anotherembodiment, the dimerization domain is that of a basic regionhelix-loop-helix (“bHLH”) protein. (See Murre et al., 1989, Cell56:777-783. See also Davis et al., 1990, Cell 60:733-746; Voronova andBaltimore, 1990, Proc. Natl. Acad. Sci. USA 87:4722-26.) Particularlyuseful hHLH proteins are myc, max, and mac.

In yet other embodiments, the dimerization domain is an immunoglobulinconstant region such as, for example, a heavy chain constant region or adomain thereof (e.g., a C_(H)1 domain, a C_(H)2 domain, and/or a C_(H)3domain). (See, e.g., U.S. Pat. Nos. 5,155,027; 5,336,603; 5,359,046; and5,349,053; EP 0 367 166; and WO 96/04388.)

Heterodimers are known to form between Fos and Jun (Bohmann et al.,1987, Science 238:1386-1392), among members of the ATF/CREB family (Haiet al., 1989, Genes Dev. 3:2083-2090), among members of the C/EBP family(Cao et al., 1991, Genes Dev. 5:1538-52; Williams et al., 1991, GenesDev. 5:1553-67; Roman et al., 1990, Genes Dev. 4:1404-15), and betweenmembers of the ATF/CREB and Fos/Jun families (Hai and Curran, 1991,Proc. Natl. Acad. Sci. USA 88:3720-24). Therefore, when a CD19 bindingagent is administered to a subject as a heterodimer comprising differentdimerization domains, any combination of the foregoing may be used.

In other embodiments, an anti-CD19 antibody derivative is an anti-CD19antibody conjugated to a second antibody (an “antibody heteroconjugate”)(see, e.g. U.S. Pat. No. 4,676,980). Heteroconjugates can be formed, forexample, between an antibody that binds to CD19 and an antibody thatbinds to a surface receptor or receptor complex that mediates ADCC,phagocytosis, and/or CDC, such as CD16/FcγRIII, CD64/FcγRI, killer cellactivating or inhibitory receptors, or the complement control proteinCD59. In one embodiment, the binding of the portion of the multispecificantibody to the second cell surface molecule or receptor complexenhances the effector functions of an anti-CD19 antibody.

Antibodies and other binding agents can be assayed for specific bindingto CD19 (e.g., human CD19) by any of various known methods. Immunoassayswhich can be used include, for example, competitive and non-competitiveassay systems. Such assays are routine and well-known in the art. (See,e.g., Ausubel et al., eds., Short Protocols in Molecular Biology (JohnWiley and Sons, Inc., New York, 4th ed. 1999); Harlow and Lane, UsingAntibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1999.))

Further, the binding affinity of a CD19 binding agent (e.g., anti-CD19antibody or derivative thereof) to CD19 and the off-rate of a bindingagent-CD19 interaction can be determined by competitive binding assays.One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled CD19 (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledCD19, and the detection of the antibody bound to the labeled CD19. Theaffinity of the antibody for CD19 and the binding off-rates can then bedetermined from the data by Scatchard plot analysis. Competition with asecond antibody can also be determined using radioimmunoassays. In thiscase, CD19 is incubated with the antibody of interest conjugated to alabeled compound (e.g., ³H or ¹²⁵I) in the presence of increasingamounts of an unlabeled second antibody. Alternatively, the bindingaffinity of an antibody to CD19 and the on- and off-rates of anantibody-CD19 interaction can be determined by surface plasmonresonance. In some embodiments, the anti-CD19 antibodies or derivativesthereof can be targeted to and accumulate on the membrane of aCD19-expressing cell.

CD19 binding agents (e.g., anti-CD19 antibody or derivative thereof) canbe produced by methods known in the art for the synthesis of proteins,typically, e.g., by recombinant expression techniques. Recombinantexpression of an antibody or derivative thereof typically involvesconstruction of an expression vector containing a nucleic acid thatencodes the binding agent. A vector for the production of the proteinmolecule may be produced by recombinant DNA technology using techniquesknown in the art. Standard techniques such as, for example, thosedescribed in Sambrook and Russell, Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,3rd ed., 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2nd ed.,1989); Short Protocols in Molecular Biology (Ausubel et al., John Wileyand Sons, New York, 4th ed., 1999); and Glick and Pasternak, MolecularBiotechnology: Principles and Applications of Recombinant DNA (ASMPress, Washington, D.C., 2nd ed., 1998) can be used for recombinantnucleic acid methods, nucleic acid synthesis, cell culture, transgeneincorporation, and recombinant protein expression.

For example, for recombinant expression of an anti-CD19 antibody, anexpression vector may encode a heavy or light chain thereof, or a heavyor light chain variable domain, operably linked to a promoter. Anexpression vector may include, for example, the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464), and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain. Theexpression vector is transferred to a host cell by conventionaltechniques, and the transfected cells are then cultured by conventionaltechniques to produce the anti-CD19 antibody. In typical embodiments forthe expression of double-chain antibodies, vectors encoding both theheavy and light chains can be co-expressed in the host cell forexpression of the entire immunoglobulin molecule.

A variety of prokaryotic and eukaryotic host-expression vector systemscan be utilized to express a CD19 binding agent (e.g., anti-CD19antibody or derivative thereof). Typically, eukaryotic cells,particularly for whole recombinant anti-CD19 antibody molecules, areused for the expression of the recombinant protein. For example,mammalian cells such as Chinese hamster ovary cells (CHO; e.g., DG44),in conjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus, is an effective expressionsystem for the production of anti-CD19 antibodies and derivativesthereof (see, e.g., Foecking et al., 1986, Gene 45:101; Cockett et al.,1990, Bio/Technology 8:2). CD19 binding aagents can also be expressedusing the CHEF system. (See, e.g., U.S. Pat. No. 5,888,809.)

Other host-expression systems include, for example, plasmid-basedexpression systems in bacterial cells (see, e.g., Ruther et al., 1983,EMBO 1,2:1791; Inouye and Inouye, 1985, Nucleic Acids Res. 13:3101-3109;Van Heeke and Schuster, 1989, J. Biol. Chem. 24:5503-5509); insectsystems such as, e.g., the use of Autographa californica nuclearpolyhedrosis virus (AcNPV) expression vector in Spodoptera frugiperdacells; and viral-based expression systems in mammalian cells, such as,e.g., adenoviral-based systems (see, e.g., Logan and Shenk, 1984, Proc.Natl. Acad. Sci. USA 81:355-359; Bittner et al., 1987, Methods inEnzymol. 153:51-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processing(e.g., glycosylation, phosphorylation, and cleavage) of the proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript and geneproduct can be used. Such mammalian host cells include, for example,CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, and W138.

A stable expression system is typically used for long-term, high-yieldproduction of a recombinant CD19 binding agent. For example, cell linesthat stably express the anti-CD19 antibody or derivative thereof can beengineered by transformation of host cells with DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites) and aselectable marker, followed by growth of the transformed cells in aselective media. The selectable marker confers resistance to theselection and allows cells to stably integrate the DNA into theirchromosomes and grow to form foci which in turn can be cloned andexpanded into cell lines. A number of selection systems can be used,including, for example, the herpes simplex virus thymidine kinase,hypoxanthineguanine phosphoribosyltransferase, and adeninephosphoribosyltransferase genes, which can be employed in tk⁻, hgprt⁻ oraprt⁻ cells, respectively. Also, antimetabolite resistance can be usedas the basis of selection for the following genes: dhfr, which confersresistance to methotrexate; gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG-418; and hygro, which confers resistance to hygromycin. Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone, and such methods aredescribed, for example, in Ausubel et al., eds., in the CurrentProtocols in Molecular Biology series of laboratory technique manuals,1987-1999 Current Protocols, © 1994-1999 John Wiley and Sons, Inc.).;Kriegler, Gene Transfer and Expression, A Laboratory Manual (StocktonPress, N.Y., 1990); Current Protocols in Human Genetics (Dracopoli etal. eds., John Wiley and Sons, N.Y., 1994, Chapters 12 and 13); andColberre-Garapin et al., 1981, J. Mol. Biol. 150:1.

The expression levels of an antibody or derivative can be increased byvector amplification. (See generally Bebbington and Hentschel, The Useof Vectors Based on Gene Amplification for the Expression of ClonedGenes in Mammalian Cells in DNA Cloning, Vol. 3 (Academic Press, NewYork, 1987).) When a marker in the vector system expressing an anti-CD19antibody or derivative thereof is amplifiable, an increase in the levelof inhibitor present in host cell culture media will select host cellsthat have increased copy number of a marker gene conferring resistanceto the inhibitor. The copy number of an associated antibody gene willalso be increased, thereby increasing expression of the antibody orderivative thereof (see, e.g., Crouse et al., 1983, Mol. Cell. Biol.3:257).

Where a CD19 binding agent comprises both a heavy and a light chain, thehost cell may be co-transfected with two expression vectors, the firstvector encoding the heavy chain protein and the second vector encodingthe light chain protein. The two vectors may contain identicalselectable markers which enable equal expression of heavy and lightchain proteins. Alternatively, a single vector may be used whichencodes, and is capable of expressing, both heavy and light chainproteins. In such situations, the light chain is typically placed beforethe heavy chain to avoid an excess of toxic free heavy chain (see, e.g.,Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA77:2197). The coding sequences for the heavy and light chains maycomprise cDNA or genomic DNA.

Once a CD19 binding agent has been produced (e.g., by an animal,chemical synthesis, or recombinant expression), it can be purified byany suitable method for purification of proteins, including, forexample, by chromatography (e.g., ion exchange or affinitychromatography (such as, for example, Protein A chromatography forpurification of antibodies having an intact Fc region)), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. An anti-CD19 antibody or derivative thereofcan, for example, be fused to a marker sequence, such as a peptide, tofacilitate purification by affinity chromatography. Suitable markeramino acid sequences include, e.g., a hexa-histidine peptide, such asthe tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, Calif.,91311), and the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767),and the “flag” tag.

Typically, the CD19 binding agent is substantially purified (e.g.,substantially free from substances that limit its effect or produceundesired side-effects). In some embodiments, the CD19 binding agent isat least about 40% pure, at least about 50% pure, or at least about 60%pure. In some embodiments, the CD19 binding agent is at least about60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-98% pure.In some embodiments, the CD19 binding agent is approximately 99% pure.

Further CD19 binding agents can include fusion proteins (i.e., proteinsthat are recombinantly fused or chemically conjugated, including bothcovalent and non-covalent conjugation) to heterologous proteins (oftypically at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or at least 100amino acids). In some embodiments, such a CD19 binding agent includesthe amino acid sequence of a humanized heavy and/or light chain variableregion that specifically binds to CD19 and optionally an immunoglobulineffector region or a functional equivalent thereof. As used herein, afunctional equivalent of an immunoglobulin effector region binds to anFc receptor on an immune cell with phagocytic or lytic activity, or theimmunoglobulin effector region binds to one or more components of thecomplement system. The linkage of the CD19 binding portion to theheterologous protein is not necessarily direct, but may occur through alinker sequence(s).

For example, a CD19 binding agent can be produced recombinantly byfusing a humanized variable region in frame with a sequence coding for aheterologous protein. The heterologous protein optionally can include aneffector region or a functional equivalent thereof and may provide oneor more of the following characteristics: promote stable expression;provide a means of facilitating high yield recombinant expression;and/or provide a multimerization domain.

A CD19 binding agent can be identified using any method suitable forscreening for protein-protein interactions. Typically, proteins areinitially identified by their ability to specifically bind to CD19.Among the traditional methods which can be employed are “interactioncloning” techniques which entail probing expression libraries withlabeled CD19 in a manner similar to the technique of antibody probing ofλgt11 libraries. By way of example and not limitation, this can beachieved as follows: a cDNA clone encoding CD19 can be modified at theC-terminus by inserting the phosphorylation site for the heart musclekinase (HMK) (see, e.g., Blanar and Rutter, 1992, Science 256:1014-18).The recombinant protein is expressed in E. coli and purified on aGDP-affinity column to homogeneity (Edery et al., 1988, Gene 74:517-25)and labeled using γ³²P-ATP and bovine heart muscle kinase (Sigma-AldrichCo., St. Louis, Mo.) to a specific activity of 1×10⁸ cpm/μg, and used toscreen a human placenta λgt11 cDNA library in a “far-Western assay”(Blanar and Rutter, 1992, Science 256:1014-18). Plaques that interactwith the CD19 probe are isolated. The cDNA inserts of positive λ plaquesare released and subcloned into a vector suitable for sequencing, suchas pBluescript KS (Stratagene, La Jolla, Calif.).

One method which detects protein interactions in vivo is the two-hybridsystem. One version of this system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA 88:9578-82) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Anti-CD19 ligand-drug conjugate compounds can include useful classes ofcytotoxic or immunomodulatory agents, for example, antitubulin agents,auristatins, DNA minor groove binders, DNA replication inhibitors,alkylating agents (e.g., platinum complexes such as cis-platin,mono(platinum), bis(platinum) and tri-nuclear platinum complexes andcarboplatin), anthracyclines, antibiotics, antifolates, antimetabolites,chemotherapy sensitizers, duocarmycins, etoposides, fluorinatedpyrimidines, ionophores, lexitropsins, nitrosoureas, platinols,pre-forming compounds, purine antimetabolites, puromycins, radiationsensitizers, steroids, taxanes, topoisomerase inhibitors, vincaalkaloids, or the like.

Chemotherapeutic agents can be used in a ligand-drug conjugate, or incombination with anti-CD19 antibodies or anti-CD19 ligand-drugconjugate, in methods for treatment of neoplastic disease. In certainembodiments, an antibody-cytotoxin conjugate comprising antibodies toCD19 can be used to boost immunity induced through standard cancertreatments. In these instances, it can be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr et al., Cancer Research 58:5301-5304, 1998). The scientific rationale behind the combined use ofCD19 antibody and chemotherapy is that cell death, that is a consequenceof the cytotoxic action of most chemotherapeutic compounds, shouldresult in increased levels of tumor antigen in the antigen presentationpathway. Thus, CD19 antibody can boost an immune response primed tochemotherapy release of tumor cells.

B cell lymphoma and leukemia, can include, but are not limited to,non-Hodgkin lymphoma, chronic lymphocytic leukemia, and acutelymphoblastic leukemia

“Leukemia” refers to progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia is generally clinically classified onthe basis of (1) the duration and character of the disease—acute orchronic; (2) the type of cell involved; myeloid (myelogenous), lymphoid(lymphogenous), or monocytic; and (3) the increase or non-increase inthe number of abnormal cells in the blood—leukemic or aleukemic(subleukemic). Leukemia includes, for example, acute nonlymphocyticleukemia, chronic lymphocytic leukemia, acute granulocytic leukemia,chronic granulocytic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, a leukocythemic leukemia,basophylic leukemia, blast cell leukemia, bovine leukemia, chronicmyelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilicleukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia,hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia,acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia,lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia,megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia,myeloblastic leukemia, myelocytic leukemia, myeloid granulocyticleukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cellleukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

Ligand-Drug Conjugate Compounds

The present invention provides, inter alia, ligand-drug conjugatecompounds for targeted delivery of drugs. The inventors have made thediscovery that the ligand-drug conjugate compounds have potent cytotoxicand/or cytostatic activity against B cells expressing CD19. Theligand-drug conjugate compounds comprise a Ligand unit covalently linkedto at least one Drug unit. The Drug units can be covalently linkeddirectly or via a Linker unit (-LU—).

In some embodiments, the ligand drug conjugate compound has thefollowing formula:

L-(LU-D)_(p)  (I)

or a pharmaceutically acceptable salt or solvate thereof; wherein:

L is the Ligand unit, i.e., CD19 binding agent of the present invention,and

(LU-D) is a Linker unit-Drug unit moiety, wherein:

LU- is a Linker unit, and

-D is a drug unit having cytostatic or cytotoxic activity against atarget cell; and

p is an integer from 1 to about 20.

In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. Inother embodiments, p is 1, 2, 3, 4, 5 or 6. In some embodiments, p is 2or 4.

In some embodiments, the ligand drug conjugate compound has thefollowing formula:

L-(A_(a)-W_(w)—Y_(y)-D)_(p)  (II)

or a pharmaceutically acceptable salt or solvate thereof;

wherein:

L is the Ligand unit, i.e. CD19 binding agent; and

-A_(a)-W_(w)—Y_(y)— is a Linker unit (LU), wherein:

-A- is a Stretcher unit,

a is 0 or 1,

each —W— is independently an Amino Acid unit,

w is an integer ranging from 0 to 12,

—Y— is a self-immolative spacer unit,

y is 0, 1 or 2;

-D is a drug units having cytostatic or cytotoxic activity against thetarget cell; and

p is an integer from 1 to about 20.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. Insome embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1. In someembodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p ranges from 2 to 8,2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is 1,2, 3, 4, 5 or 6. In some embodiments, p is 2 or 4. In some embodiments,when w is not zero, y is 1 or 2.

The drug loading is represented by p, the average number of drugmolecules per Ligand, e.g., antibody in a molecule. Drug loading mayrange from 1 to 20 drugs (D) per Ligand. The average number of drugs perligand in preparation of conjugation reactions may be characterized byconventional means such as mass spectroscopy, ELISA assay, and HPLC. Thequantitative distribution of Ligand-Drug-Conjugates in terms of p mayalso be determined. In some instances, separation, purification, andcharacterization of homogeneous Ligand-Drug-conjugates where p is acertain value from Ligand-Drug-Conjugates with other drug loadings maybe achieved by means such as reverse phase HPLC or electrophoresis. Inexemplary embodiments, p is from 2 to 8.

The generation of ligand-drug conjugate compounds can be accomplished byany technique known to the skilled artisan. Briefly, the ligand-drugconjugate compounds comprise a CD19 binding agent as the ligand unit, adrug, and optionally a linker that joins the drug and the binding agent.A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is often accomplished byreaction of the amino acid residues of the binding agent, e.g., antibodymolecule, including the amine groups of lysine, the free carboxylic acidgroups of glutamic and aspartic acid, the sulfhydryl groups of cysteineand the various moieties of the aromatic amino acids. One of the mostcommonly used non-specific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule. Alsoavailable for attachment of drugs to binding agents is the Schiff basereaction. This method involves the periodate oxidation of a drug thatcontains glycol or hydroxy groups, thus forming an aldehyde which isthen reacted with the binding agent. Attachment occurs via formation ofa Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In certain embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with theCD19 binding agent under appropriate conditions.

Each of the particular units of the ligand-drug conjugate compounds isdescribed in more detail herein. The synthesis and structure of linkerunits, stretcher units, amino acid units, self-immolative spacer unit,and drug units are also described in U.S. Patent Application PublicationNos. 2003-0083263, 2005-0238649 and 2005-0009751, each if which isincorporated herein by reference in its entirety and for all purposes.

Linker Units

Typically, the ligand-drug conjugate compounds comprise a linker regionbetween the drug unit and the ligand unit. In some embodiments, thelinker is cleavable under intracellular conditions, such that cleavageof the linker releases the drug unit from the ligand in theintracellular environment.

For example, in some embodiments, the linker is cleavable by a cleavingagent that is present in the intracellular environment (e.g., within alysosome or endosome or caveolea). The linker can be, e.g., a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease.Typically, the peptidyl linker is at least two amino acids long or atleast three amino acids long. Cleaving agents can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123).Most typical are peptidyl linkers that are cleavable by enzymes that arepresent in CD19-expressing cells. For example, a peptidyl linker that iscleavable by the thiol-dependent protease cathepsin-B, which is highlyexpressed in cancerous tissue, can be used (e.g., a Phe-Leu or aGly-Phe-Leu-Gly linker). Other such linkers are described, e.g., in U.S.Pat. No. 6,214,345, incorporated herein by reference in its entirety andfor all purposes. In a specific embodiment, the peptidyl linkercleavable by an intracellular protease is a Val-Cit linker or a Phe-Lyslinker (see, e.g., U.S. Pat. No. 6,214,345, which describes thesynthesis of doxorubicin with the val-cit linker). One advantage ofusing intracellular proteolytic release of the therapeutic agent is thatthe agent is typically attenuated when conjugated and the serumstabilities of the conjugates are typically high.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-S-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931;Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935.)

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

Typically, the linker is not substantially sensitive to theextracellular environment. As used herein, “not substantially sensitiveto the extracellular environment,” in the context of a linker, meansthat no more than about 20%, typically no more than about 15%, moretypically no more than about 10%, and even more typically no more thanabout 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of ligand-drug conjugate compound, are cleaved whenthe ligand-drug conjugate compound presents in an extracellularenvironment (e.g., in plasma). Whether a linker is not substantiallysensitive to the extracellular environment can be determined, forexample, by incubating with plasma the ligand-drug conjugate compoundfor a predetermined time period (e.g., 2, 4, 8, 16, or 24 hours) andthen quantitating the amount of free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (i.e.,in the milieu of the linker-therapeutic agent moiety of the ligand-drugconjugate compound as described herein). In yet other embodiments, thelinker promotes cellular internalization when conjugated to both theauristatin compound and the anti-CD19 antibody.

A variety of linkers that can be used with the present compositions andmethods are described in WO 2004010957 entitled “Drug Conjugates andTheir Use for Treating Cancer, An Autoimmune Disease or an InfectiousDisease” filed Jul. 31, 2003, and U.S. Publication No. 2006/0074008entitled “Drug Conjugates and their use for treating cancer, anautoimmune disease or an infectious disease”, filed Jul. 31, 2002 (thedisclosure of which is incorporated by reference herein in its entiretyand for all purposes).

A “Linker unit” (LU) is a bifunctional compound that can be used to linka Drug unit and a Ligand unit to form a ligand-drug conjugate compound.In some embodiments, the Linker unit has the formula:

-A_(a)-W_(w)—Y_(y)—

wherein:-A- is a Stretcher unit,

a is 0 or 1,

each —W— is independently an Amino Acid unit,

w is an integer ranging from 0 to 12,

—Y— is a self-immolative Spacer unit, and

y is 0, 1 or 2.

In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1 or 2. Insome embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or 1.

The Stretcher Unit

The Stretcher unit (A), when present, is capable of linking a Ligandunit to an Amino Acid unit (—W—), if present, to a Spacer unit (—Y—), ifpresent; or to a Drug unit (-D). Useful functional groups that can bepresent on a CD19 binding agent, either naturally or via chemicalmanipulation include, but are not limited to, sulfhydryl, amino,hydroxyl, the anomeric hydroxyl group of a carbohydrate, and carboxyl.Suitable functional groups are sulfhydryl and amino. In one example,sulfhydryl groups can be generated by reduction of the intramoleculardisulfide bonds of an anti-CD19 antibody. In another embodiment,sulfhydryl groups can be generated by reaction of an amino group of alysine moiety of an anti-CD19 antibody with 2-iminothiolane (Traut'sreagent) or other sulfhydryl generating reagents. In certainembodiments, the anti-CD19 antibody is a recombinant antibody and isengineered to carry one or more lysines. In certain other embodiments,the recombinant anti-CD19 antibody is engineered to carry additionalsulfhydryl groups, e.g., additional cysteines.

In one embodiment, the Stretcher unit forms a bond with a sulfur atom ofthe Ligand unit. The sulfur atom can be derived from a sulfhydryl groupof a Ligand. Representative Stretcher units of this embodiment aredepicted within the square brackets of Formulas IIIa and IIIb, whereinL-, —W—, —Y—, -D, w and y are as defined above, and R₁₇ is selected from—C₁-C₁₀ alkylene-, —C₁-C₁₀ alkenylene-, —C₁-C₁₀ alkynylene-,carbocyclo-, —O—(C₁-C₈ alkylene)-, O—(C₁-C₈ alkenylene)-, —O—(C₁-C₈alkynylene)-, -arylene-, alkylene-arylene-, —C₂-C₁₀ alkenylene-arylene,—C₂-C₁₀ alkynylene-arylene, -arylene-C₁-C₁₀ alkylene-, -arylene-C₂-C₁₀alkenylene-, -arylene-C₂-C₁₀ alkynylene-, —C₁-C₁₀alkylene-(carbocyclo)-, —C₂-C₁₀ alkenylene-(carbocyclo)-, —C₂-C₁₀alkynylene-(carbocyclo)-, -(carbocyclo)-C₁-C₁₀ alkylene-,-(carbocyclo)-C₂-C₁₀ alkenylene-, -(carbocyclo)-C₂-C₁₀ alkynylene,-heterocyclo-, alkylene-(heterocyclo)-, —C₂-C₁₀alkenylene-(heterocyclo)-, —C₂-C₁₀ alkynylene-(heterocyclo)-,-(heterocyclo)-C₁-C₁₀ alkylene-, -(heterocyclo)-C₂-C₁₀ alkenylene-,-(heterocyclo)-C₁-C₁₀ alkynylene-, —(CH₂CH₂O)_(r)—, or—(CH₂CH₂O)_(r)—CH₂—, and r is an integer ranging from 1-10, wherein saidalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,carbocyle, carbocyclo, heterocyclo, and arylene radicals, whether aloneor as part of another group, are optionally substituted. Alkylene,alkenylene, alkynylene radicals, whether alone or as part of anothergroup, can be optionally substituted with, for example, one or moregroups independently selected from A1; carbocyclo radicals, whetheralone or as part of another group, can be optionally substituted with,for example, one or more groups independently selected from A2; aryleneradicals, whether alone or as part of another group, can be optionallysubstituted with, for example, one or more groups independently selectedfrom A3; heterocyclo radicals, whether alone or as part of anothergroup, can be optionally substituted with, for example, one or moregroups independently selected from A4. A1, A2, A3, and A4 are as definedherein. It is to be understood from all the exemplary embodiments thateven where not denoted expressly, from 1 to 20 drug moieties can belinked to a Ligand (p=1-20).

An illustrative Stretcher unit is that of Formula Ma wherein R¹⁷ is—(CH₂)₅—:

Another illustrative Stretcher unit is that of Formula IIIa wherein R¹⁷is —(CH₂CH₂O)_(r)—CH₂—; and r is 2:

Still another illustrative Stretcher unit is that of Formula Mb whereinR¹⁷ is —(CH₂)₅—:

In certain embodiments, the Stretcher unit is linked to the Ligand unitvia a disulfide bond between a sulfur atom of the Ligand unit and asulfur atom of the Stretcher unit. A representative Stretcher unit ofthis embodiment is depicted within the square brackets of Formula IV,wherein R¹⁷, L-, —W—, —Y—, -D, w and y are as defined above.

L-SS—R¹⁷—C(O)W_(w)—Y_(y)-D   IV

It should be noted that throughout this application, the S moiety in theformula below refers to a sulfur atom of the Ligand unit, unlessotherwise indicated by context.

In yet other embodiments, the Stretcher contains a reactive site thatcan form a bond with a primary or secondary amino group of a Ligand.Examples of these reactive sites include, but are not limited to,activated esters such as succinimide esters, 4 nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted withinthe square brackets of Formulas Va and Vb, wherein —R¹⁷—, L-, —W—, —Y—,-D, w and y are as defined above;

In some embodiments, the Stretcher contains a reactive site that isreactive to a modified carbohydrate's (—CHO) group that can be presenton a Ligand. For example, a carbohydrate can be mildly oxidized using areagent such as sodium periodate and the resulting (—CHO) unit of theoxidized carbohydrate can be condensed with a Stretcher that contains afunctionality such as a hydrazide, an oxime, a primary or secondaryamine, a hydrazine, a thiosemicarbazone, a hydrazine carboxylate, and anarylhydrazide such as those described by Kaneko et al., 1991,Bioconjugate Chem. 2:133-41. Representative Stretcher units of thisembodiment are depicted within the square brackets of Formulas VIa, VIb,and VIc, wherein —R¹⁷—, L-, —W—, —Y—, -D, w and y are as defined asabove.

The Amino Acid Unit

The Amino Acid unit (—W—), when present, links the Stretcher unit to theSpacer unit if the Spacer unit is present, links the Stretcher unit tothe Drug moiety if the Spacer unit is absent, and links the Ligand unitto the Drug unit if the Stretcher unit and Spacer unit are absent.

W_(w)— can be, for example, a dipeptide, tripeptide, tetrapeptide,pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide,decapeptide, undecapeptide or dodecapeptide unit. Each —W— unitindependently has the formula denoted below in the square brackets, andw is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH₂,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

In some embodiments, the Amino Acid unit can be enzymatically cleaved byone or more enzymes, including a cancer or tumor-associated protease, toliberate the Drug unit (-D), which in one embodiment is protonated invivo upon release to provide a Drug (D).

In certain embodiments, the Amino Acid unit can comprise natural aminoacids. In other embodiments, the Amino Acid unit can comprisenon-natural amino acids. Illustrative Ww units are represented byformulas (VII)-(IX):

wherein R²⁰ and R²¹ are as follows:

(VIII)

R²⁰ R²¹ Benzyl (CH₂)₄NH₂; methyl (CH₂)₄NH₂; isopropyl (CH₂)₄NH₂;isopropyl (CH₂)₃NHCONH₂; benzyl (CH₂)₃NHCONH₂; isobutyl (CH₂)₃NHCONH₂;sec-butyl (CH₂)₃NHCONH₂;

(CH₂)₃NHCONH₂; benzyl methyl; benzyl (CH₂)₃NHC(═NH)NH₂;wherein R²⁰, R²¹ and R²² are as follows:

(IX)

R²⁰ R²¹ R²² benzyl benzyl (CH₂)₄NH₂; isopropyl benzyl (CH₂)₄NH₂; and Hbenzyl (CH₂)₄NH₂;wherein R²⁰, R²¹, R²² and R²³ are as follows:

R²⁰ R²¹ R²² R²³ H benzyl isobutyl H; and methyl isobutyl methylisobutyl.

Exemplary Amino Acid units include, but are not limited to, units offormula VII where: R²⁰ is benzyl and R²¹ is —(CH₂)₄NH₂; R²⁰ is isopropyland R²¹ is —(CH₂)₄NH₂; or R²⁰ is isopropyl and R²¹ is —(CH₂)₃NHCONH₂.Another exemplary Amino Acid unit is a unit of formula VII wherein R²⁰is benzyl, R²¹ is benzyl, and R²² is —(CH₂)₄NH₂.

Useful —W_(w)— units can be designed and optimized in their selectivityfor enzymatic cleavage by a particular enzyme, for example, atumor-associated protease. In one embodiment, a —W_(w)— unit is thatwhose cleavage is catalyzed by cathepsin B, C and D, or a plasminprotease.

In one embodiment, —W_(w)— is a dipeptide, tripeptide, tetrapeptide orpentapeptide. When R¹⁹, R²⁰, R²¹, R²² or R²³ is other than hydrogen, thecarbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached is chiral.

Each carbon atom to which R¹⁹, R²⁰, R²¹, R²² or R²³ is attached isindependently in the (S) or (R) configuration.

In one aspect of the Amino Acid unit, the Amino Acid unit isvaline-citrulline (vc or val-cit). In another aspect, the Amino Acidunit is phenylalanine-lysine (i.e., fk). In yet another aspect of theAmino Acid unit, the Amino Acid unit is N-methylvaline-citrulline. Inyet another aspect, the Amino Acid unit is 5-aminovaleric acid, homophenylalanine lysine, tetraisoquinolinecarboxylate lysine,cyclohexylalanine lysine, isonepecotic acid lysine, beta-alanine lysine,glycine serine valine glutamine and isonepecotic acid.

The Spacer Unit

The Spacer unit (—Y—), when present, links an Amino Acid unit to theDrug unit when an Amino Acid unit is present. Alternately, the Spacerunit links the Stretcher unit to the Drug unit when the Amino Acid unitis absent. The Spacer unit also links the Drug unit to the Ligand unitwhen both the Amino Acid unit and Stretcher unit are absent.

Spacer units are of two general types: non self-immolative orself-immolative. A non self-immolative Spacer unit is one in which partor all of the Spacer unit remains bound to the Drug moiety aftercleavage, particularly enzymatic, of an Amino Acid unit from theligand-drug conjugate. Examples of a non self-immolative Spacer unitinclude, but are not limited to a (glycine-glycine) Spacer unit and aglycine Spacer unit (both depicted in Scheme 1) (infra). When aconjugate containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via an enzyme (e.g., a tumor-cellassociated-protease, a cancer-cell-associated protease or alymphocyte-associated protease), a glycine-glycine-Drug moiety or aglycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, anindependent hydrolysis reaction takes place within the target cell,cleaving the glycine-Drug moiety bond and liberating the Drug.

In some embodiments, a non self-immolative Spacer unit (—Y—) is -Gly-.In some embodiments, a non self-immolative Spacer unit (—Y—) is-Gly-Gly-.

In one embodiment, a Drug-Linker conjugate is provided in which theSpacer unit is absent (y=0), or a pharmaceutically acceptable salt orsolvate thereof.

Alternatively, a conjugate containing a self-immolative Spacer unit canrelease -D. As used herein, the term “self-immolative Spacer” refers toa bifunctional chemical moiety that is capable of covalently linkingtogether two spaced chemical moieties into a stable tripartite molecule.It will spontaneously separate from the second chemical moiety if itsbond to the first moiety is cleaved.

In some embodiments, —Y_(y)— is a p-aminobenzyl alcohol (PAB) unit (seeSchemes 2 and 3) whose phenylene portion is substituted with Q_(m)wherein Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or-cyano; and m is an integer ranging from 0-4. The akyl, alkenyl andalkynyl groups, whether alone or as part of another group, can beoptionally substituted with R1 as defined herein.

In some embodiments, —Y— is a PAB group that is linked to —W_(w)— viathe amino nitrogen atom of the PAB group, and connected directly to -Dvia a carbonate, carbamate or ether group. Without being bound by anyparticular theory or mechanism, Scheme 2 depicts a possible mechanism ofDrug release of a PAB group which is attached directly to -D via acarbamate or carbonate group as described by Toki et al., 2002, J. Org.Chem. 67:1866-1872.

In Scheme 2, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 20. The akyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted with A1 as definedherein.

Without being bound by any particular theory or mechanism, Scheme 3depicts a possible mechanism of Drug release of a PAB group which isattached directly to -D via an ether or amine linkage, wherein Dincludes the oxygen or nitrogen group that is part of the Drug unit.

In Scheme 3, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; and p ranges from 1to about 20. The akyl, alkenyl and alkynyl groups, whether alone or aspart of another group, can be optionally substituted with A1 as definedherein.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al., 1999,Bioorg. Med. Chem. Lett. 9:2237) and ortho or para-aminobenzylacetals.Spacers can be used that undergo cyclization upon amide bond hydrolysis,such as substituted and unsubstituted 4-aminobutyric acid amides(Rodrigues et al., 1995, Chemistry Biology 2:223), appropriatelysubstituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm etal., 1972, J. Amer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acidamides (Amsberry et al., 1990, J. Org. Chem. 55:5867). Elimination ofamine-containing drugs that are substituted at the α-position of glycine(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples ofself-immolative spacers.

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4, whichcan be used to incorporate and release multiple drugs.

In Scheme 4, Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl,—O—(C₁-C₈ alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen,-nitro or -cyano; m is an integer ranging from 0-4; n is 0 or 1; and pranges raging from 1 to about 20. The akyl, alkenyl and alkynyl groups,whether alone or as part of another group, can be optionally substitutedwith A1 as defined herein.

In some embodiments, the -D moieties are the same. In yet anotherembodiment, the -D moieties are different.

In one aspect, Spacer units (—Y_(y)—) are represented by Formulas(X)-(XII):

wherein Q is —C₁-C₈ alkyl, —C₁-C₈ alkenyl, —C₁-C₈ alkynyl, —O—(C₁-C₈alkyl), —O—(C₁-C₈ alkenyl), —O—(C₁-C₈ alkynyl), -halogen, -nitro or-cyano; and m is an integer ranging from 0-4. The akyl, alkenyl andalkynyl groups, whether alone or as part of another group, can beoptionally substituted with A1 as defined herein.

Embodiments of the Formula I and II comprising Ligand-drug conjugatecompounds can include:

wherein w and y are each 0, 1 or 2,

-   -   and,

wherein w and y are each 0,

The Drug Unit

The drug moiety (D) can be any cytotoxic, cytostatic or immunomodulatory(e.g., immunosuppressive) or drug. D is a Drug unit (moiety) having anatom that can form a bond with the Spacer unit, with the Amino Acidunit, with the Stretcher unit or with the Ligand unit. In someembodiments, the Drug unit D has a nitrogen atom that can form a bondwith the Spacer unit. As used herein, the terms “drug unit” and “drugmoiety” are synonymous and used interchangeably.

Useful classes of cytotoxic or immunomodulatory agents include, forexample, antitubulin agents, auristatins, DNA minor groove binders, DNAreplication inhibitors, alkylating agents (e.g., platinum complexes suchas cis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like.

Individual cytotoxic or immunomodulatory agents include, for example, anandrogen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine,bleomycin, busulfan, buthionine sulfoximine, calicheamicin,camptothecin, carboplatin, carmustine (BSNU), CC-1065, chlorambucil,cisplatin, colchicine, cyclophosphamide, cytarabine, cytidinearabinoside, cytochalasin B, dacarbazine, dactinomycin (formerlyactinomycin), daunorubicin, decarbazine, docetaxel, doxorubicin,etoposide, an estrogen, 5-fluordeoxyuridine, 5-fluorouracil,gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), maytansine, mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA,topotecan, vinblastine, vincristine, vinorelbine, VP-16 and VM-26.

In some typical embodiments, suitable cytotoxic agents include, forexample, DNA minor groove binders (e.g., enediynes and lexitropsins, aCBI compound; see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes(e.g., paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065,SN-38, topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epothilone A and B, estramustine, cryptophysins, cemadotin,maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

In some embodiments, the Drug is an anti-tubulin agent. Examples ofanti-tubulin agents include, but are not limited to, taxanes (e.g.,Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vincaalkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).Other antitubulin agents include, for example, baccatin derivatives,taxane analogs (e.g., epothilone A and B), nocodazole, colchicine andcolcimid, estramustine, cryptophycins, cemadotin, maytansinoids,combretastatins, discodermolide, and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., 1992, Cancer Res. 52:127-131).

In some embodiments, the Drug is an auristatin, such as auristatin E(also known in the art as dolastatin-10) or a derivative thereof.Typically, the auristatin E derivative is, e.g., an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatin derivatives include AFP,MMAF, and MMAE. The synthesis and structure of auristatin derivativesare described in U.S. Patent Application Publication Nos. 2003-0083263,2005-0238649 and 2005-0009751; International Patent Publication No. WO04/010957, International Patent Publication No. WO 02/088172, and U.S.Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860;5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284;5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744;4,879,278; 4,816,444; and 4,486,414, each of which is incorporated byrefernence herein in its entirety and for all purposes.

Auristatins have been shown to interfere with microtubule dynamics andnuclear and cellular division and have anticancer activity. Auristatinsof the present invention bind tubulin and can exert a cytotoxic orcytostatic effect on a CD19 expressing cell line. There are a number ofdifferent assays, known in the art, that can be used for determiningwhether an auristatin or resultant antibody-drug conjugate exerts acytostatic or cytotoxic effect on a desired cell line, see e.g., Example7.

Methods for determining whether a compound binds tubulin are known inthe art. See, for example, Muller et al., Anal. Chem 2006, 78,4390-4397; Hamel et al., Molecular Pharmacology, 1995 47: 965-976; andHamel et al., The Journal of Biological Chemistry, 1990 265:28,17141-17149. For purposes of the present invention, the relativeaffinity of a compound to tubulin can be determined. Preferredauristatins of the present invention bind tubulin with an affinityranging from 10 fold lower that the binding affinity of MMAE to tubulinto 10 fold, 20 fold or even 100 fold higher than the binding affinity ofMMAE to tublin.

In some embodiments, -D is an auristatin of the formula D_(E), D_(F) orD_(Z):

or a pharmaceutically acceptable salt or solvate form thereof;wherein, independently at each location:

R² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R³ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

R⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

R⁵ is H or C₁-C₈ alkyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(s)— wherein R^(a) and R^(b) are independently H, C₁-C₂₀alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, or carbocycle and s is 2, 3, 4, 5or 6,

R⁶ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R⁷ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle);

each R⁸ is independently H, OH, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, —O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₁-C₂₀ alkynyl), orcarbocycle;

R⁹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

R²⁴ is aryl, heterocycle, or carbocycle;

R²⁵ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carbocycle,—O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₂-C₂₀ alkynyl), or OR¹⁸wherein R¹⁸ is H, a hydroxyl protecting group, or a direct bond whereOR¹⁸ represents ═O;

R²⁶ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl, aryl,heterocycle, or carbocycle;

R¹⁰ is aryl or heterocycle;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, orC₂-C₂₀ alkynyl;

R¹¹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), —C₂-C₂₀alkynylene(heterocycle) —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—C_(H)(R¹⁵)₂;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₂₀ alkylene, C₂-C₂₀ alkenylene, or C₂-C₂₀ alkynylene;

R¹⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl;

each occurrence of R¹⁶ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH;

n is an integer ranging from 0 to 6;

R²⁷ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, O—(C₁-C₂₀alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₂-C₂₀ alkynyl), halogen, —NO₂, —COOH,or —C(O)OR²⁸ wherein R²⁸ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, aryl, heterocycle, —(CH₂CH₂O)_(r)—H, —(CH₂CH₂O)_(r)—CH₃, or—(CH₂CH₂O)_(r)—CH₂CH₂C(O)OH; wherein r is an integer ranging from 1-10;and

X is —(CR²⁹ ₂)_(I)— wherein R²⁹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl and I is an integer ranging from 0 to 10; wherein saidalkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,carbocyle, and heterocycle radicals, whether alone or as part of anothergroup, are optionally substituted.

Auristatins of the formula D_(E), D_(F) or D_(Z) include those wherein

R² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl, each of which isoptionally substituted with one or more groups independently selectedfrom A1;

R³ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆carbocycle, —C₁-C₂₀ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀alkenylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclicC₃-C₆ carbocycle), C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀alkenylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle,—C₁-C₂₀ alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or—C₂-C₂₀ alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,alkylene, alkenylene, and alkynylene radicals whether alone or as partof another group are optionally substituted with one or more groupsindependently selected from A1, said carbocycle radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A2, said aryl radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A3, and said heterocycle radicalswhether alone or as part of another group are optionally substitutedwith one or more groups independently selected from A4;

R⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆carbocycle, —C₁-C₂₀ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀alkenylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclicC₃-C₆ carbocycle), C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀alkenylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle,—C₁-C₂₀ alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or—C₂-C₂₀ alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,alkylene, alkenylene, and alkynylene radicals whether alone or as partof another group are optionally substituted with one or more groupsindependently selected from A1, said carbocycle radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A2, said aryl radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A3, and said heterocycle radicalswhether alone or as part of another group are optionally substitutedwith one or more groups independently selected from A4;

R⁵ is H or C₁-C₈ alkyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(s)— wherein R^(a) and R^(b) are independently H, C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or carbocycle, and s is 2, 3, 4, 5or 6;

R⁶ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl, wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1;

R⁷ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆carbocycle, —C₁-C₂₀ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀alkenylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclicC₃-C₆ carbocycle), C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀alkenylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle,—C₁-C₂₀ alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or—C₂-C₂₀ alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,alkylene, alkenylene, and alkynylene radicals whether alone or as partof another group are optionally substituted with one or more groupsindependently selected from A1, said carbocycle radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A2, said aryl radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A3, and said heterocycle radicalswhether alone or as part of another group are optionally substitutedwith one or more groups independently selected from A4;

each R⁸ is independently H, OH, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀alkynyl, —O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₁-C₂₀ alkynyl), orcarbocycle, wherein said alkyl, alkenyl, and alkynyl radicals, whetheralone or as part of another group, are optionally substituted with oneor more groups independently selected from A1 and said carbocycle isoptionally substituted with one or more groups independently selectedfrom A2;

R⁹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; wherein saidalkyl, alkenyl and alkynyl radical are optionally substituted with oneor more groups independently selected from A1;

R²⁴ is aryl, heterocycle, or carbocycle; wherein said carbocycle radicalis optionally substituted with one or more groups independently selectedfrom A2, said aryl radical is optionally substituted with one or moregroups independently selected from A3, and said heterocycle radical isoptionally substituted with one or more groups independently selectedfrom A4;

R²⁵ is selected from H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,carbocycle, OH, —O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₂-C₂₀alkynyl) or OR¹⁸; wherein said alkyl, alkenyl, and alkynyl radicals,whether alone or as part of another group, are optionally substitutedwith one or more groups independently selected from A1, and saidcarbocycle is optionally substituted with one or more groupsindependently selected from A2;

R¹⁸ is H, a hydroxyl protecting group, or a direct bond where OR¹⁸represents ═O;

R²⁶ is selected from H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, orcarbocycle; wherein said alkyl, alkenyl, and alkynyl radicals areoptionally substituted with one or more groups independently selectedfrom A1, and said carbocycle radical is optionally substituted with oneor more groups independently selected from A2;

R¹⁰ is aryl optionally substituted with one or more groups independentlyselected from A3, or heterocycle optionally substituted with one or moregroups independently selected from A4;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, orC₂-C₂₀ alkynyl, each of which is optionally substituted with one or moregroups independently selected from A1;

R¹¹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, carbocycle,—C₁-C₂₀ alkylene (carbocycle), —C₂-C₂₀ alkenylene(carbocycle), —C₂-C₂₀alkynylene(carbocycle), aryl, —C₁-C₂₀ alkylene(aryl), —C₂-C₂₀alkenylene(aryl), —C₂-C₂₀ alkynylene(aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), —C₂-C₂₀alkynylene(heterocycle) —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—C_(H)(R¹⁵)₂wherein said alkyl, alkenyl and alkynyl radicals are optionallysubstituted with one or more groups independently selected from A1, saidcarbocycle radical is optionally substituted with one or more groupsindependently selected from A2, said aryl radical is optionallysubstituted with one or more groups independently selected from A3, andsaid heterocycle is optionally substituted with one or more groupsindependently selected from A4;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₂₀ alkylene, C₂-C₂₀ alkenylene, or C₂-C₂₀ alkynylene, each ofwhich is optionally substituted with one or more groups independentlyselected from A1;

R¹⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl wherein said alkyl, alkenyland alkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

each occurrence of R¹⁶ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH wherein said alkyl, alkenyl andalkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

n is an integer ranging from 0 to 6;

R²⁷ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, O—(C₁-C₂₀alkyl), —O—(C₂-C₂₀ alkenyl), —O—(C₂-C₂₀ alkynyl), halogen, —NO₂, —COOH,or —C(O)OR²⁸ wherein said alkyl, alkenyl and alkynyl radicals areoptionally substituted with one or more groups independently selectedfrom A1 and R²⁸ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,aryl, heterocycle, —(CH₂CH₂O)_(r)—H, —(CH₂CH₂O)_(r)—CH₃, or—(CH₂CH₂O)_(r)—CH₂CH₂C(O)OH; wherein r is an integer ranging from 1-10and wherein said alkyl, alkenyl and alkynyl radicals are optionallysubstituted with one or more groups independently selected from A1; saidaryl radical is optionally substituted with one or more groupsindependently selected from A3; and said heterocycle radical isoptionally substituted with one or more groups independently selectedfrom A4; and

X is —(CR²⁹ ₂)_(I)— wherein R²⁹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl and I is an integer ranging from 0 to 10 and wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1;

A1 is halogen, optionally substituted —O—(C₁-C₈ alkyl), optionallysubstituted —O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈alkynyl), optionally substituted -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′,—C(O)NH₂, —C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′,—S(O)R′, —OH, ═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ isindependently selected from H, optionally substituted —C₁-C₈ alkyl,optionally substituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈alkynyl, or optionally substituted aryl, and wherein said optionallysubstituted O—(C₁-C₈ alkyl), optionally substituted —O—(C₂-C₈ alkenyl),optionally substituted —O—(C₂-C₈ alkynyl), optionally substituted aryl,optionally substituted C₁-C₈ alkyl, optionally substituted —C₂-C₈alkenyl, and optionally substituted —C₂-C₈ alkynyl groups can beoptionally substituted with one or more groups independently selectedfrom —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen, —O—(C₁-C₈alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl, —C(O)R″,—OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂—NHC(O)R″, —SR″,—SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″), —N(R″)₂ and —CN,where each R″ is independently selected from H, —C₁-C₈ alkyl, —C₂-C₈alkenyl, —C₂-C₈ alkynyl, or aryl;

A2 is halogen, optionally substituted C₁-C₈ alkyl, optionallysubstituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈ alkynyl,optionally substituted —O—(C₁-C₈ alkyl), optionally substituted—O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈ alkynyl),optionally substituted aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,═O, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from H, optionally substituted —C₁-C₈ alkyl, optionallysubstituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈ alkynyl, oroptionally substituted aryl and wherein said optionally substituted—C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkenyl, optionallysubstituted —C₂-C₈ alkynyl, optionally substituted —O—(C₁-C₈ alkyl),optionally substituted —O—(C₂-C₈ alkenyl), optionally substituted—O—(C₂-C₈ alkynyl), and optionally substituted aryl groups can beoptionally substituted with one or more substituents independentlyselected from C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂—NHC(O)R″,—SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″), —N(R″)₂ and—CN, where each R″ is independently selected from H, —C₁-C₈ alkyl,—C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl;

A3 is halogen, optionally substituted —C₁-C₈ alkyl, optionallysubstituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈ alkynyl,optionally substituted —O—(C₁-C₈ alkyl), optionally substituted—O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈ alkynyl),optionally substituted -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,—NO₂, —N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ isindependently selected from H, optionally substituted —C₁-C₈ alkyl,optionally substituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈alkynyl, or optionally substituted aryl and wherein said optionallysubstituted —C₁-C₈ alkyl, optionally substituted —C₂-C₈ alkenyl,optionally substituted —C₂-C₈ alkynyl, optionally substituted O—(C₁-C₈alkyl), optionally substituted —O—(C₂-C₈ alkenyl), optionallysubstituted —O—(C₂-C₈ alkynyl), and optionally substituted aryl, groupscan be further optionally substituted with one or more substituentsindependently selected from C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl,halogen, —O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl),-aryl, —C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″,—C(O)N(R″)₂—NHC(O)R″, —SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂,—NH(R″), —N(R″)₂ and —CN, where each R″ is independently selected fromH, —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl;

and A4 is optionally substituted —C₁-C₈ alkyl, optionally substituted—C₂-C₈ alkenyl, optionally substituted —C₂-C₈ alkynyl, halogen,optionally substituted —O—(C₁-C₈ alkyl), optionally substituted—O—(C₂-C₈ alkenyl), optionally substituted —O—(C₂-C₈ alkynyl),optionally substituted -aryl, —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)NH₂,—C(O)NHR′, —C(O)N(R′)₂, —NHC(O)R′, —SR′, —SO₃R′, —S(O)₂R′, —S(O)R′, —OH,—N₃, —NH₂, —NH(R′), —N(R′)₂ and —CN, where each R′ is independentlyselected from H, optionally substituted —C₁-C₈ alkyl, optionallysubstituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈ alkynyl, oroptionally substituted aryl and wherein said optionally substitutedO—(C₁-C₈ alkyl), optionally substituted —O—(C₂-C₈ alkenyl), optionallysubstituted —O—(C₂-C₈ alkynyl), optionally substituted —C₁-C₈ alkyl,optionally substituted —C₂-C₈ alkenyl, optionally substituted —C₂-C₈alkynyl, and optionally substituted aryl groups can be furtheroptionally substituted with one or more substituents independentlyselected from —C₁-C₈ alkyl, —C₂-C₈ alkenyl, —C₂-C₈ alkynyl, halogen,—O—(C₁-C₈ alkyl), —O—(C₂-C₈ alkenyl), —O—(C₂-C₈ alkynyl), -aryl,—C(O)R″, —OC(O)R″, —C(O)OR″, —C(O)NH₂, —C(O)NHR″, —C(O)N(R″)₂—NHC(O)R″,—SR″, —SO₃R″, —S(O)₂R″, —S(O)R″, —OH, —N₃, —NH₂, —NH(R″), —N(R″)₂ and—CN, where each R″ is independently selected from H, —C₁-C₈ alkyl,—C₂-C₈ alkenyl, —C₂-C₈ alkynyl, or aryl; or a pharmaceuticallyacceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted.

Auristatins of the formula D_(E) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are unsubstituted and the groups of R²⁴,R²⁵ and R²⁶ are optionally substituted as described herein.

Auristatins of the formula D_(E) include those wherein

R² is C₁-C₂₀ alkyl optionally substituted with one or more groupsindependently selected from A1;

R³ and R⁷ are independently selected from H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene radicals whether alone or as part of anothergroup are optionally substituted with one or more groups independentlyselected from A1, said carbocycle radicals whether alone or as part ofanother group are optionally substituted with one or more groupsindependently selected from A2, said aryl radicals whether alone or aspart of another group are optionally substituted with one or more groupsindependently selected from A3, and said heterocycle radicals whetheralone or as part of another group are optionally substituted with one ormore groups independently selected from A4;

R⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆carbocycle, —C₁-C₂₀ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀alkenylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclicC₃-C₆ carbocycle), C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀alkenylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle,—C₁-C₂₀ alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or—C₂-C₂₀ alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,alkylene, alkenylene, and alkynylene radicals whether alone or as partof another group are optionally substituted with one or more groupsindependently selected from A1, said carbocycle radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A2, said aryl radicals whether aloneor as part of another group are optionally substituted with one or moregroups independently selected from A3, and said heterocycle radicalswhether alone or as part of another group are optionally substitutedwith one or more groups independently selected from A4;

R⁵ is H or C₁-C₈ alkyl;

or R⁴ and R⁵ jointly form a carbocyclic ring and have the formula—(CR^(a)R^(b))_(s)— wherein R^(a) and R^(b) are independently selectedfrom H, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, or carbocycle, and sis selected from 2, 3, 4, 5 or 6;

R⁶ is C₁-C₂₀ alkyl optionally substituted with one or more groupsindependently selected from A1;

each R⁸ is independently selected from OH, O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀alkenyl), or —O—(C₂-C₂₀ alkynyl) wherein said alkyl, alkenyl, andalkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

R⁹ is hydrogen or C₁-C₂₀ alkyl optionally substituted with one or moregroups independently selected from A1;

R²⁴ is aryl, heterocycle, or carbocycle; wherein said carbocycle radicalis optionally substituted with one or more groups independently selectedfrom A2, said aryl radical is optionally substituted with one or moregroups independently selected from A3, and said heterocycle radical isoptionally substituted with one or more groups independently selectedfrom A4;

R²⁵ is OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is selected from H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, orcarbocycle; wherein said alkyl, alkenyl, and alkynyl radicals areoptionally substituted with one or more groups independently selectedfrom A1, and said carbocycle radical is optionally substituted with oneor more groups independently selected from A2;

and A1, A2, A3, and A4 are as defined herein; or a pharmaceuticallyacceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein

R² is C₁-C₈ alkyl;

R³, R⁴ and R⁷ are independently selected from H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene radicals whether alone or as part of anothergroup are optionally substituted with one or more groups independentlyselected from A1, said carbocycle radicals whether alone or as part ofanother group are optionally substituted with one or more groupsindependently selected from A2, said aryl radicals whether alone or aspart of another group are optionally substituted with one or more groupsindependently selected from A3, and said heterocycle radicals whetheralone or as part of another group are optionally substituted with one ormore groups independently selected from A4;

R⁵ is hydrogen;

R⁶ is C₁-C₈ alkyl;

each R⁸ is independently selected from OH, O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀alkenyl), or —O—(C₂-C₂₀ alkynyl) wherein said alkyl, alkenyl, andalkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

R⁹ is hydrogen or C₁-C₈ alkyl;

R²⁴ is phenyl optionally substituted with one or more groupsindependently selected from A3;

R²⁵ is OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is selected from H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, orcarbocycle; wherein said alkyl, alkenyl, and alkynyl radicals areoptionally substituted with one or more groups independently selectedfrom A1, and said carbocycle radical is optionally substituted with oneor more groups independently selected from A2; and

A1, A2, A3, and A4 are as defined herein; or a pharmaceuticallyacceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein

R² is methyl;

R³ is H, C₁-C₈ alkyl, C₂-C₈ alkenyl, or C₂-C₈ alkynyl, wherein saidalkyl, alkenyl and alkynyl radicals are optionally optionallysubstituted with one or more groups independently selected from A1;

R⁴ is H, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, monocyclic C₃-C₆carbocycle, C₆-C₁₀ aryl, —C₁-C₈ alkylene(C₆-C₁₀ aryl), C₂-C₈alkenylene(C₆-C₁₀ aryl), —C₂-C₈ alkynylene(C₆-C₁₀ aryl), —C₁-C₈ alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₈ alkenylene (monocyclic C₃-C₆carbocycle), —C₂-C₈ alkynylene(monocyclic C₃-C₆ carbocycle); whereinsaid alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynyleneradicals whether alone or as part of another group are optionallysubstituted with one or more groups independently selected from A1; saidcarbocyle radicals whether alone or as part of another group areoptionally substituted with one or more groups independently selectedfrom A2; and said aryl radicals whether alone or as part of anothergroup are optionally substituted with one or more groups independentlyselected from A3;

R⁵ is H; R⁶ is methyl;

R⁷ is C₁-C₈ alkyl, C₂-C₈ alkenyl or C₂-C₈ alkynyl;

each R⁸ is methoxy;

R⁹ is hydrogen or C₁-C₈ alkyl;

R²⁴ is phenyl;

R²⁵ is OR¹⁸; wherein R¹⁸ is H, a hydroxyl protecting group, or a directbond where OR¹⁸ represents ═O;

R²⁶ is methyl; and A1, A2, and A3 are as defined herein; or apharmaceutically acceptable salt or solvate form thereof.

Auristatins of the formula D_(E) include those wherein

R² is methyl; R³ is H or C₁-C₃ alkyl; R⁴ is C₁-C₅ alkyl; R⁵ is H; R⁶ ismethyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is hydrogen orC₁-C₈ alkyl; R²⁴ is phenyl; R²⁵ is OR¹⁸; wherein R¹⁸ is H, a hydroxylprotecting group, or a direct bond where OR¹⁸ represents ═O; and R²⁶ ismethyl; or a pharmaceutically acceptable salt or solvate form thereof.

Auristatins of the formula D_(F) or D_(Z) include those wherein

R² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl, each of which isoptionally substituted with one or more groups independently selectedfrom A1;

R³, R⁴, and R⁷ are independently selected from H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene radicals whether alone or as part of anothergroup are optionally substituted with one or more groups independentlyselected from A1, said carbocycle radicals whether alone or as part ofanother group are optionally substituted with one or more groupsindependently selected from A2, said aryl radicals whether alone or aspart of another group are optionally substituted with one or more groupsindependently selected from A3, and said heterocycle radicals whetheralone or as part of another group are optionally substituted with one ormore groups independently selected from A4;

R⁵ is H;

R⁶ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl, wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1;

each R⁸ is independently selected from H, OH, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, —O—(C₁-C₂₀ alkyl), —O—(C₂-C₂₀ alkenyl),—O—(C₁-C₂₀ alkynyl), or carbocycle, wherein said alkyl, alkenyl, andalkynyl radicals, whether alone or as part of another group, areoptionally substituted with one or more groups independently selectedfrom A1 and said carbocycle is optionally substituted with one or moregroups independently selected from A2;

R⁹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; wherein saidalkyl, alkenyl and alkynyl radical are optionally substituted with oneor more groups independently selected from A1;

R¹⁰ is phenyl optionally substituted with one or more groupsindependently selected from A3;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, orC₂-C₂₀ alkynyl, each of which is optionally substituted with one or moregroups independently selected from A1;

R¹¹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, aryl,heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂ wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1, said aryl radical isoptionally substituted with one or more groups independently selectedfrom A3, and said heterocycle is optionally substituted with one or moregroups independently selected from A4;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₂₀ alkylene, C₂-C₂₀ alkenylene, or C₂-C₂₀ alkynylene, each ofwhich is optionally substituted with one or more groups independentlyselected from A1;

R¹⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl wherein said alkyl, alkenyland alkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

each occurrence of R¹⁶ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH wherein said alkyl, alkenyl andalkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

n is an integer ranging from 0 to 6;

R²⁷ is H; and

X is —(CR²⁹ ₂)_(I)— wherein I is 0; and A1, A2, A3, and A4 are asdefined herein; or a pharmaceutically acceptable salt or solvate formthereof.

Auristatins of the formula D_(F) or D_(Z) include those wherein

R² is methyl;

R³, R⁴, and R⁷ are independently selected from H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, monocyclic C₃-C₆ carbocycle, —C₁-C₂₀alkylene(monocyclic C₃-C₆ carbocycle), —C₂-C₂₀ alkenylene(monocyclicC₃-C₆ carbocycle), —C₂-C₂₀ alkynylene(monocyclic C₃-C₆ carbocycle),C₆-C₁₀ aryl, —C₁-C₂₀ alkylene(C₆-C₁₀ aryl), —C₂-C₂₀ alkenylene(C₆-C₁₀aryl), —C₂-C₂₀ alkynylene(C₆-C₁₀ aryl), heterocycle, —C₁-C₂₀alkylene(heterocycle), —C₂-C₂₀ alkenylene(heterocycle), or —C₂-C₂₀alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene radicals whether alone or as part of anothergroup are optionally substituted with one or more groups independentlyselected from A1, said carbocycle radicals whether alone or as part ofanother group are optionally substituted with one or more groupsindependently selected from A2, said aryl radicals whether alone or aspart of another group are optionally substituted with one or more groupsindependently selected from A3, and said heterocycle radicals whetheralone or as part of another group are optionally substituted with one ormore groups independently selected from A4;

R⁵ is H;

R⁶ is methyl;

each R⁸ is methoxy;

R⁹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl; wherein saidalkyl, alkenyl and alkynyl radical are optionally substituted with oneor more groups independently selected from A1;

R¹⁰ is aryl optionally substituted with one or more groups independentlyselected from A3, or heterocycle optionally substituted with one or moregroups independently selected from A4;

Z is O, S, NH, or NR¹², wherein R¹² is C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, orC₂-C₂₀ alkynyl, each of which is optionally substituted with one or moregroups independently selected from A1;

R¹¹ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, aryl,heterocycle, —(R¹³O)_(m)—R¹⁴, or —(R¹³O)_(m)—CH(R¹⁵)₂ wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1, said aryl radical isoptionally substituted with one or more groups independently selectedfrom A3, and said heterocycle is optionally substituted with one or moregroups independently selected from A4;

m is an integer ranging from 1-1000;

R¹³ is C₂-C₂₀ alkylene, C₂-C₂₀ alkenylene, or C₂-C₂₀ alkynylene, each ofwhich is optionally substituted with one or more groups independentlyselected from A1;

R¹⁴ is H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, or C₂-C₂₀ alkynyl wherein saidalkyl, alkenyl and alkynyl radicals are optionally substituted with oneor more groups independently selected from A1;

each occurrence of R¹⁵ is independently H, COOH, —(CH₂)_(n)—N(R¹⁶)₂,—(CH₂)_(n)—SO₃H, —(CH₂)_(n)—SO₃—C₁-C₂₀ alkyl, —(CH₂)_(n)—SO₃—C₂-C₂₀alkenyl, or —(CH₂)_(n)—SO₃—C₂-C₂₀ alkynyl wherein said alkyl, alkenyland alkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

each occurrence of R¹⁶ is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl,C₂-C₂₀ alkynyl or —(CH₂)_(n)—COOH wherein said alkyl, alkenyl andalkynyl radicals are optionally substituted with one or more groupsindependently selected from A1;

n is an integer ranging from 0 to 6;

R²⁷ is H; and

X is —(CR²⁹ ₂)_(I)— wherein I is 0; and A1, A2, A3, and A4 are asdefined herein; or a pharmaceutically acceptable salt or solvate formthereof.

In certain of these embodiments, R¹⁰ is phenyl optionally substitutedwith one or more groups independently selected from A3.

Auristatins of the formula D_(F) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ are unsubstituted and the groups of R¹⁰and R¹¹ are as described herein.

Auristatins of the formula D_(F) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted

Auristatins of the formula D_(F) include those wherein

R² is methyl; R³ is H or C₁-C₃ alkyl; R⁴ is C₁-C₅ alkyl; R⁵ is H; R⁶ ismethyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹ is hydrogen orC₁-C₈ alkyl; R¹⁰ is phenyl optionally substituted with one or moregroups independently selected from A3; Z is O, S, or NH; R¹¹ is asdefined herein; or a pharmaceutically acceptable salt or solvate formthereof.

Auristatins of the formula D_(F) include those wherein

R¹ is hydrogen; R² is methyl; R³ is H or C₁-C₃ alkyl; R⁴ is C₁-C₅ alkyl;R⁵ is H; R⁶ is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹is hydrogen or C₁-C₈ alkyl; R¹⁰ is phenyl; Z is O or NH; R¹¹ is asdefined herein; or a pharmaceutically acceptable salt or solvate formthereof.

Auristatins of the formula D_(Z) include those wherein the groups of R²,R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R²⁷, R²⁸, and R²⁹ are unsubstituted and thegroups of R¹⁰ and R¹¹ are as described herein.

Auristatins of the formula D_(Z) include those wherein said alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl, carbocyle,and heterocycle radicals are unsubstituted

Auristatins of the formula D_(Z) include those wherein

R¹ is hydrogen; R² is methyl; R³ is H or C₁-C₃ alkyl; R⁴ is C₁-C₅ alkyl;R⁵ is H; R⁶ is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹is hydrogen or C₁-C₈ alkyl;

R¹⁰ is phenyl optionally substituted with one or more groupsindependently selected from A3; Z is O, S, or NH; R¹¹ is as definedherein; R²⁷ is H; and X is —(CR²⁹ ₂)_(I)— wherein I is 0; or apharmaceutically acceptable salt or solvate form thereof.

Auristatins of the formula D_(Z) include those wherein

R¹ is hydrogen; R² is methyl; R³ is H or C₁-C₃ alkyl; R⁴ is C₁-C₅ alkyl;R⁵ is H; R⁶ is methyl; R⁷ is isopropyl or sec-butyl; R⁸ is methoxy; R⁹is hydrogen or C₁-C₈ alkyl; R¹⁰ is phenyl; R¹¹ is as defined herein; R²⁷is H; X is —(CR²⁹ ₂)_(I)— wherein I is 0; and Z is O or NH; or apharmaceutically acceptable salt or solvate form thereof.

Auristatins of the formula D_(Z) include those wherein R¹¹ is—(CH₂CH₂O)_(r)—H, —(CH₂CH₂O)_(r)—CH₃, or —(CH₂CH₂)_(r)—CH₂CH₂C(O)OH;wherein r is an integer ranging from 1-10; or a pharmaceuticallyacceptable salt or solvate form thereof.

Auristatins of the formula D_(Z) include those wherein the phenyl groupat the amino terminus is para substituted as shown below:

Auristatins of the formula D_(E), D_(F) or D_(Z) include those whereinR³, R⁴ and R⁷ are independently isopropyl or sec-butyl and R⁵ is —H. Inan exemplary embodiment, R³ and R⁴ are each isopropyl, R⁵ is H, and R⁷is sec-butyl. The remainder of the substituents are as defined herein.

Auristatins of the formula D_(E), D_(F) or D_(Z) include those whereinR² and R⁶ are each methyl, and R⁹ is H. The remainder of thesubstituents are as defined herein.

Auristatins of the formula D_(E), D_(F) or D_(Z) include those whereineach occurrence of R⁸ is —OCH₃. The remainder of the substituents are asdefined herein.

Auristatins of the formula D_(E), D_(F) or D_(Z) include those whereinR³ and R⁴ are each isopropyl, R² and R⁶ are each methyl, R⁵ is H, R⁷ issec-butyl, each occurrence of R⁸ is —OCH₃, and R⁹ is H. The remainder ofthe substituents are as defined herein.

Auristatins of the formula D_(F) or D_(Z) include those wherein Z is —O—or —NH—. The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) or D_(Z) include those wherein R¹⁰ isaryl. The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) or D_(Z) include those wherein, R¹⁰ is-phenyl. The remainder of the substituents are as defined herein.

Auristatins of the formula D_(F) or D_(Z) include those wherein Z is—O—, and R¹¹ is H, methyl or t-butyl. The remainder of the substituentsare as defined herein.

Auristatins of the formula D_(F) or D_(Z) include those wherein, when Zis —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is —(CH₂)_(n)—N(R¹⁶)₂, and R¹⁶ is—C₁-C₈ alkyl or —(CH₂)_(n)—COOH. The remainder of the substituents areas defined herein.

Auristatins of the formula D_(F) or D_(Z) include those wherein when Zis —NH, R¹¹ is —CH(R¹⁵)₂, wherein R¹⁵ is —(CH₂)_(n)—SO₃H. The remainderof the substituents are as defined herein.

Illustrative Drug units (-D) include the drug units having the followingstructures:

or pharmaceutically acceptable salts or solvates thereof.

In one aspect, hydrophilic groups, such as but not limited totriethylene glycol esters (TEG) can be attached to the Drug Unit at R¹¹.Without being bound by theory, the hydrophilic groups assist in theinternalization and non-agglomeration of the Drug Unit.

In some embodiments, the Drug unit is not TZT-1027. In some embodiments,the Drug unit is not auristatin E, dolastatin 10, or auristatin PE.

Exemplary ligand-drug conjugate compounds have the following structureswherein “mAb-s-” represents an anti-CD19 antibody:

or pharmaceutically acceptable salt or solvate forms thereof, whereinVal is valine, and Cit is citrulline.

In certain embodiments, the Drug is an antimetabolite. Theantimetabolite can be, for example, a purine antagonist (e.g.,azothioprine or mycophenolate mofetil), a dihydrofolate reductaseinhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine,vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

In other embodiments, the Drug is tacrolimus, cyclosporine or rapamycin.In further embodiments, the Drug is aldesleukin, alemtuzumab,alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenictrioxide, bexarotene, bexarotene, calusterone, capecitabine, celecoxib,cladribine, Darbepoetin alfa, Denileukin diftitox, dexrazoxane,dromostanolone propionate, epirubicin, Epoetin alfa, estramustine,exemestane, Filgrastim, floxuridine, fludarabine, fulvestrant,gemcitabine, gemtuzumab ozogamicin, goserelin, idarubicin, ifosfamide,imatinib mesylate, Interferon alfa-2a, irinotecan, letrozole,leucovorin, levamisole, meclorethamine or nitrogen mustard, megestrol,mesna, methotrexate, methoxsalen, mitomycin C, mitotane, nandrolonephenpropionate, oprelvekin, oxaliplatin, pamidronate, pegademase,pegaspargase, pegfilgrastim, pentostatin, pipobroman, plicamycin,porfimer sodium, procarbazine, quinacrine, rasburicase, Rituximab,Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide,testolactone, thioguanine, toremifene, Tositumomab, Trastuzumab,tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,vinorelbine and zoledronate.

In some embodiments, the Drug moiety is an immunomodulatory agent. Theimmunomodulatory agent can be, for example, gancyclovir, etanercept,tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine,mycophenolate mofetil or methotrexate. Alternatively, theimmunomodulatory agent can be, for example, a glucocorticoid (e.g.,cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisoneor dexamethasone).

In some embodiments, the immunomodulatory agent is an anti-inflammatoryagent, such as arylcarboxylic derivatives, pyrazole-containingderivatives, oxicam derivatives and nicotinic acid derivatives. Classesof anti-inflammatory agents include, for example, cyclooxygenaseinhibitors, 5-lipoxygenase inhibitors, and leukotriene receptorantagonists.

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,ibuprofen, indomethacin, ketoprofen, nabumetone, naproxen, sulindac,tenoxicam, tolmetin, and acetylsalicylic acid.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, Iampalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, Ionapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl) sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SK&F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

In certain embodiments, the cytotoxic or cytostatic agent is adolastatin. In certain embodiments, the cytotoxic or cytostatic agent isof the auristatin class. Thus, in a specific embodiment, the cytotoxicor cytostatic agent is MMAE (Formula XI). In another specificembodiment, the cytotoxic or cytostatic agent is AFP (Formula XVI).

In certain embodiments, the cytotoxic or cytostatic agent is a compoundof formulas XII-XXI or pharmaceutically acceptable salt or solvate formthereof:

Methods of determining whether a Drug or Ligand-Drug conjugate exerts acytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of a Ligand Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theLigand Drug conjugate in a cell culture medium; culturing the cells fora period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Ligand Drug conjugate.

For determining whether a Ligand Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of ³H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of ³H-thymidine into cells of the culture is measured inthe presence and absence of the Ligand Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that a Ligand Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

The effects of Ligand Drug conjugates can be tested or validated inanimal models. A number of established animal models of cancers areknown to the skilled artisan, any of which can be used to assay theefficacy of a Ligand Drug conjugate. Non-limiting examples of suchmodels are described infra. Moreover, small animal models to examine thein vivo efficacies of Ligand Drug conjugates can be created byimplanting human tumor cell lines into appropriate immunodeficientrodent strains, e.g., athymic nude mice or SCID mice.

Ligand Unit

The Ligand unit (L) has at least one functional group that can form abond with a functional group of a Linker unit. Useful functional groupsthat can be present on a Ligand unit, either naturally, via chemicalmanipulation or via engineering, include, but are not limited to,sulfhydryl (—SH), amino, hydroxyl, carboxy, the anomeric hydroxyl groupof a carbohydrate, and carboxyl. In some embodiments, a Ligand unitfunctional group is a sulfhydryl group. The sulfhydryl group istypically a solvent accessible sulfhydryl group, such as a solventaccessible sulfhydryl group on a cysteine residue. Sulfhydryl groups canbe generated by reduction of an intramolecular or intermoleculardisulfide bond of a Ligand. Sulfhydryl groups also can be generated byreaction of an amino group of a lysine moiety of a Ligand using2-iminothiolane (Traut's reagent) or another sulfhydryl generatingreagent.

In some embodiments, one or more sulfhydryl groups are engineered into aLigand unit, such as by amino acid substitution. For example, asulfhydryl group can be introduced into a Ligand unit. In someembodiments, a sulfhydryl group is introduced by an amino acidsubstitution of serine or threonine to a cysteine residue, and/or byaddition of a cysteine residue into a Ligand unit (an engineeredcysteine residue). In some embodiments, the cysteine residue is aninternal cysteine residue, i.e., not located at the N-terminus orC-terminus of the Ligand moiety.

In an exemplary embodiment, a cysteine residue can be engineered into anantibody heavy or light variable region (e.g., of an antibody fragment,such as a diabody) by amino acid substitution. The amino acidsubstitution is typically introduced into the framework region and islocated distal to the epitope-binding face of the variable region. Forexample, the amino acid substitution can be at least 10 angstroms, atleast 20 angstroms or at least 25 angstroms from the epitope-bindingface or the CDRs. Suitable positions for substitution of a cysteineresidue can be determined based on the known or predicted threedimensional structures of antibody variable regions. (See generallyHolliger and Hudson, 2005, Nature BioTechnology 23(9):1126-1136.) Inexemplary embodiments, a serine to cysteine amino acid substitution isintroduced at amino acid position 84 of the V_(H) region and/or position14 of the V_(L) region (according to the numbering system of Kabat etal., Sequences of Proteins of Immunological Interest, 5th edition,(Bethesda, Md., NIH) 1991).

To control the number of Drug or Linker unit-Drug units attached to aLigand unit, one or more cysteine residues can be eliminated by aminoacid substitution. For example, the number of solvent accessiblecysteine residues in an immunoglobulin hinge region can be reduced byamino acid substitution of cysteine to serine residues.

In some embodiments, a Ligand unit contains 1, 2, 3, 4, 5, 6 7 or 8solvent-accessible cysteine residues. In some embodiments, a Ligand unitcontains 2 or 4 solvent-accessible cysteine residues.

Compositions and Methods of Administration

The CD19 binding agents and ligand-drug conjugate compounds can be inany form that allows for the compound to be administered to a patientfor treatment of a CD19-associated disorder. For example, the compoundcan be in the form of a liquid or solid. Typical routes ofadministration include, without limitation, parenteral, topical, oral,sublingual, rectal, vaginal, ocular, intra-tumor, and intranasal.Parenteral administration includes subcutaneous injections, intravenous,intramuscular, intrasternal injection or infusion techniques. In oneaspect, the compositions are administered parenterally. In yet anotheraspect, the compounds are administered intravenously.

Pharmaceutical compositions can be formulated so as to allow a compoundto be bioavailable upon administration of the composition to a patient.Compositions can take the form of one or more dosage units, where forexample, a tablet can be a single dosage unit.

Materials used in preparing the pharmaceutical compositions can benon-toxic in the amounts used. It will be evident to those of ordinaryskill in the art that the optimal dosage of the active ingredient(s) inthe pharmaceutical composition will depend on a variety of factors.Relevant factors include, without limitation, the type of animal (e.g.,human), the particular form of the compound, the manner ofadministration, and the composition employed.

The pharmaceutically acceptable carrier or vehicle can be particulate,so that the compositions are, for example, in tablet or powder form. Thecarrier(s) can be liquid, with the compositions being, for example, anoral syrup or injectable liquid.

When intended for oral administration, the composition is preferably insolid or liquid form, where semi-solid, semi-liquid, suspension and gelforms are included within the forms considered herein as either solid orliquid.

As a solid composition for oral administration, the composition can beformulated into a powder, granule, compressed tablet, pill, capsule,chewing gum, wafer or the like form. Such a solid composition typicallycontains one or more inert diluents. In addition, one or more of thefollowing can be present: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, or gelatin; excipients such asstarch, lactose or dextrins, disintegrating agents such as alginic acid,sodium alginate, Primogel, corn starch; lubricants such as magnesiumstearate or Sterotex; glidants such as colloidal silicon dioxide;sweetening agents such as sucrose or saccharin, a flavoring agent suchas peppermint, methyl salicylate or orange flavoring, and a coloringagent.

When the composition is in the form of a capsule, e.g., a gelatincapsule, it can contain, in addition to materials of the above type, aliquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e.g., an elixir, syrup,solution, emulsion or suspension. The liquid can be useful for oraladministration or for delivery by injection. When intended for oraladministration, a composition can comprise one or more of a sweeteningagent, preservatives, dye/colorant and flavor enhancer. In a compositionfor administration by injection, one or more of a surfactant,preservative, wetting agent, dispersing agent, suspending agent, buffer,stabilizer and isotonic agent can also be included.

The liquid compositions, whether they are solutions, suspensions orother like form, can also include one or more of the following: sterilediluents such as water for injection, saline solution, preferablyphysiological saline, Ringer's solution, isotonic sodium chloride, fixedoils such as synthetic mono or digylcerides which can serve as thesolvent or suspending medium, polyethylene glycols, glycerin,cyclodextrin, propylene glycol or other solvents; antibacterial agentssuch as benzyl alcohol or methyl paraben; antioxidants such as ascorbicacid or sodium bisulfate; chelating agents such asethylenediaminetetraacetic acid; buffers such as amino acids, acetates,citrates or phosphates; detergents, such as nonionic surfactants,polyols; and agents for the adjustment of tonicity such as sodiumchloride or dextrose. A parenteral composition can be enclosed inampoule, a disposable syringe or a multiple-dose vial made of glass,plastic or other material. Physiological saline is an exemplaryadjuvant. An injectable composition is preferably sterile.

The amount of the compound that is effective in the treatment of aparticular disorder or condition will depend on the nature of thedisorder or condition, and can be determined by standard clinicaltechniques. In addition, in vitro or in vivo assays can optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the compositions will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances.

The compositions comprise an effective amount of a compound such that asuitable dosage will be obtained. Typically, this amount is at leastabout 0.01% of a compound by weight of the composition. When intendedfor oral administration, this amount can be varied to range from about0.1% to about 80% by weight of the composition. In one aspect, oralcompositions can comprise from about 4% to about 50% of the compound byweight of the composition. In yet another aspect, present compositionsare prepared so that a parenteral dosage unit contains from about 0.01%to about 2% by weight of the compound.

For intravenous administration, the composition can comprise from about0.01 to about 100 mg of a compound per kg of the animal's body weight.In one aspect, the composition can include from about 1 to about 100 mgof a compound per kg of the animal's body weight. In another aspect, theamount administered will be in the range from about 0.1 to about 25mg/kg of body weight of a compound.

Generally, the dosage of a compound administered to a patient istypically about 0.01 mg/kg to about 100 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered to a patient isbetween about 0.01 mg/kg to about 15 mg/kg of the subject's body weight.In some embodiments, the dosage administered to a patient is betweenabout 0.1 mg/kg and about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered to a patient is between about 0.1mg/kg and about 20 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 0.1 mg/kg to about5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's bodyweight. In some embodiments, the dosage administered is between about 1mg/kg to about 15 mg/kg of the subject's body weight. In someembodiments, the dosage administered is between about 1 mg/kg to about10 mg/kg of the subject's body weight. In some embodiments, the dosageadministered is between about 0.1 to 4 mg/kg, even more preferably 0.1to 3.2 mg/kg, or even more preferably 0.1 to 2.7 mg/kg of the subject'sbody weight over a treatment cycle.

The compound or compositions can be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa). Administration can be systemic or local. Variousdelivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, and can be used to administer acompound. In certain embodiments, more than one compounds or compositionis administered to a patient.

In specific embodiments, it can be desirable to administer one or morecompounds or compositions locally to the area in need of treatment. Thiscan be achieved, for example, and not by way of limitation, by localinfusion during surgery; topical application, e.g., in conjunction witha wound dressing after surgery; by injection; by means of a catheter; bymeans of a suppository; or by means of an implant, the implant being ofa porous, non-porous, or gelatinous material, including membranes, suchas sialastic membranes, or fibers. In one embodiment, administration canbe by direct injection at the site (or former site) of a cancer, tumoror neoplastic or pre-neoplastic tissue. In another embodiment,administration can be by direct injection at the site (or former site)of a manifestation of B cell disease such as, for example, a malignancyand/or B-cell lineage malignancies.

In certain embodiments, it can be desirable to introduce one or morecompounds or compositions into the central nervous system by anysuitable route, including intraventricular and intrathecal injection.Intraventricular injection can be facilitated by an intraventricularcatheter, for example, attached to a reservoir, such as an Ommayareservoir.

In yet another embodiment, the compound or compositions can be deliveredin a controlled release system, such as but not limited to, a pump orvarious polymeric materials can be used. In yet another embodiment, acontrolled-release system can be placed in proximity of the target ofthe compound or compositions, e.g., the brain, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer(1990, Science 249:1527-1533) can be used.

The term “carrier” refers to a diluent, adjuvant or excipient, withwhich a compound is administered. Such pharmaceutical carriers can beliquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineraloil, sesame oil. The carriers can be saline, gum acacia, gelatin, starchpaste, talc, keratin, colloidal silica, urea. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used. Inone embodiment, when administered to a patient, the compound orcompositions and pharmaceutically acceptable carriers are sterile. Wateris an exemplary carrier when the compounds are administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers also includeexcipients such as starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol. The present compositions, if desired, can also containminor amounts of wetting or emulsifying agents, or pH buffering agents.

The present compositions can take the form of solutions, suspensions,emulsion, tablets, pills, pellets, capsules, capsules containingliquids, powders, sustained-release formulations, suppositories,emulsions, suspensions, or any other form suitable for use. Otherexamples of suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin, Mack PublishingCompany, Easton, Pa., 18^(th) Edition, 1995.

In an embodiment, the compounds are formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to animals, particularly human beings.Typically, the carriers or vehicles for intravenous administration aresterile isotonic aqueous buffer solutions. Where necessary, thecompositions can also include a solubilizing agent. Compositions forintravenous administration can optionally comprise a local anestheticsuch as lignocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where compound is tobe administered by infusion, it can be dispensed, for example, with aninfusion bottle containing sterile pharmaceutical grade water or saline.Where the compound is administered by injection, an ampoule of sterilewater for injection or saline can be provided so that the ingredientscan be mixed prior to administration.

Compositions for oral delivery can be in the form of tablets, lozenges,aqueous or oily suspensions, granules, powders, emulsions, capsules,syrups, or elixirs, for example. Orally administered compositions cancontain one or more optionally agents, for example, sweetening agentssuch as fructose, aspartame or saccharin; flavoring agents such aspeppermint, oil of wintergreen, or cherry; coloring agents; andpreserving agents, to provide a pharmaceutically palatable preparation.Moreover, where in tablet or pill form, the compositions can be coatedto delay disintegration and absorption in the gastrointestinal tractthereby providing a sustained action over an extended period of time.Selectively permeable membranes surrounding an osmotically activedriving compound are also suitable for orally administered compounds. Inthese later platforms, fluid from the environment surrounding thecapsule is imbibed by the driving compound, which swells to displace theagent or agent composition through an aperture. These delivery platformscan provide an essentially zero order delivery profile as opposed to thespiked profiles of immediate release formulations. A time-delay materialsuch as glycerol monostearate or glycerol stearate can also be used.

The compositions can be intended for topical administration, in whichcase the carrier can be in the form of a solution, emulsion, ointment orgel base. If intended for transdermal administration, the compositioncan be in the form of a transdermal patch or an iontophoresis device.Topical formulations can comprise a concentration of a compound of fromabout 0.05% to about 50% w/v (weight per unit volume of composition), inanother aspect, from 0.1% to 10% w/v.

The composition can include various materials that modify the physicalform of a solid or liquid dosage unit. For example, the composition caninclude materials that form a coating shell around the activeingredients. The materials that form the coating shell are typicallyinert, and can be selected from, for example, sugar, shellac, and otherenteric coating agents. Alternatively, the active ingredients can beencased in a gelatin capsule.

Whether in solid or liquid form, the present compositions can include apharmacological agent used in the treatment of cancer, e.g., B-celllineage malignancies.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

CD19-Associated Disorders

The CD19 binding agents described herein, as well as ligand-drugconjugate compounds, can be useful for treating or preventing aCD19-expressing cancer or an immunological disorder characterized byexpression or overexpression of CD19. Such expression of CD19 can be dueto, for example, increased CD19 protein levels on the cell surfaceand/or altered antigenicity of the expressed CD19. Treatment orprevention of the immunological disorder, according to the methodsdescribed herein, is achieved by administering to a subject in need ofsuch treatment or prevention an effective amount of the CD19 bindingagent or ligand-drug conjugate compound. In preferred embodiments, theligand-drug conjugate will (i) bind to activated immune cells thatexpress CD19 and that are associated with the disease state and (ii)exert a cytotoxic, cytostatic, or immunomodulatory effect on theactivated immune cells.

Immunological diseases that are characterized by inappropriateactivation of immune cells and that can be treated or prevented by themethods described herein can be classified, for example, by the type(s)of hypersensitivity reaction(s) that underlie the disorder. Thesereactions are typically classified into four types: anaphylacticreactions, cytotoxic (cytolytic) reactions, immune complex reactions, orcell-mediated immunity (CMI) reactions (also referred to as delayed-typehypersensitivity (DTH) reactions). (See, e.g., Fundamental Immunology,William E. Paul ed., Raven Press, N.Y., 3rd ed. 1993.)

Specific examples of such immunological diseases include, but are notlimited to, rheumatoid arthritis, multiple sclerosis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Grave's disease, glomerulonephritis, autoimmune hepatologicaldisorder, autoimmune inflammatory bowel disease, anaphylaxis, allergicreaction, Sjogren's syndrome, juvenile onset (Type I) diabetes mellitus,primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia,inflammatory bowel disease, polymyositis, dermatomyositis, multipleendocrine failure, Schmidt's syndrome, autoimmune uveitis, Addison'sdisease, adrenalitis, thyroiditis, Hashimoto's thyroiditis, autoimmunethyroid disease, pernicious anemia, gastric atrophy, chronic hepatitis,lupoid hepatitis, atherosclerosis, presenile dementia, demyelinatingdiseases, subacute cutaneous lupus erythematosus, hypoparathyroidism,Dressler's syndrome, autoimmune thrombocytopenia, idiopathicthrombocytopenic purpura, hemolytic anemia, pemphigus vulgaris,pemphigus, dermatitis herpetiformis, alopecia arcata, pemphigoid,scleroderma, progressive systemic sclerosis, CREST syndrome (calcinosis,Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, andtelangiectasia), adult onset diabetes mellitus (Type II diabetes), maleand female autoimmune infertility, ankylosing spondolytis, ulcerativecolitis, Crohn's disease, mixed connective tissue disease, polyarteritisnedosa, systemic necrotizing vasculitis, juvenile onset rheumatoidarthritis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, allergicencephalomyelitis, toxic epidermal necrolysis, Alport's syndrome,alveolitis, allergic alveolitis, fibrosing alveolitis, interstitial lungdisease, erythema nodosum, pyoderma gangrenosum, transfusion reaction,leprosy, malaria, leishmaniasis, trypanosomiasis, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, human immunodeficiency virus infection, echovirus infection,cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virusinfection, post vaccination syndromes, congenital rubella infection,Eaton-Lambert syndrome, relapsing polychondritis, cryoglobulinemia,Waldenstrom's macroglobulemia, Epstein-Barr virus infection, mumps,Evan's syndrome, and autoimmune gonadal failure.

Accordingly, the methods described herein encompass treatment ofdisorders of B lymphocytes (e.g., systemic lupus erythematosus,Goodpasture's syndrome, rheumatoid arthritis, and type I diabetes),Th₁-lymphocytes (e.g., rheumatoid arthritis, multiple sclerosis,psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave'sdisease, primary biliary cirrhosis, Wegener's granulomatosis,tuberculosis, or graft versus host disease), or Th₂-lymphocytes (e.g.,atopic dermatitis, systemic lupus erythematosus, atopic asthma,rhinoconjunctivitis, allergic rhinitis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th₁-lymphocytes or Th₂-lymphocytes.

The invention also includes treatment of an autoimmune disease, forexample an autoimmune disease that is mediated at least in part by Bcells. Examples of autoimmune diseases include acute necrotizinghemorrhagic leukoencephalitis; Addison's disease; Agammaglobulinemia;Allergic asthma; Allergic rhinitis; Alopecia areata; Amyloidosis;Ankylosing spondylitis; Anti-GBM/Anti-TBM nephritis; Antiphospholipidsyndrome; Autoimmune aplastic anemia; Autoimmune dysautonomia;Autoimmune hepatitis; Autoimmune hyperlipidemia; Autoimmuneimmunodeficiency; Autoimmune inner ear disease; Autoimmune myocarditis;Autoimmune thrombocytopenic purpura; Axonal & neuronal neuropathies;Balo disease; Behcet's disease; Bullous pemphigoid; Cardiomyopathy;Castleman disease; Celiac sprue (nontropical); Chagas disease; Chronicfatigue syndrome; Chronic inflammatory demyelinating polyneuropathy;Churg-Strauss syndrome; Cicatricial pemphigoid/benign mucosalpemphigoid; Crohn's disease; Cogans syndrome; Cold agglutinin disease;Congenital heart block; Coxsackie myocarditis; CREST disease; Essentialmixed cryoglobulinemia; Demyelinating neuropathies; Dermatomyositis;Devic disease; Discoid lupus; Dressler's syndrome; Endometriosis;Eosinophilic fasciitis; Erythema nodosum; Experimental allergicencephalomyelitis; Evans syndrome; Fibromyalgia; Fibrosing alveolitis;Giant cell arteritis (temporal arteritis); Goodpasture's syndrome;Graves' disease; Guillain-Barre syndrome; Hashimoto's disease; Hemolyticanemia; Henoch-Schonlein purpura; Herpes gestationis;Hypogammaglobulinemia; Idiopathic thrombocytopenic purpura; IgAnephropathy; Immunoregulatory lipoproteins; Inclusion body myositis;Insulin-dependent diabetes (type1); Interstitial cystitis; Juvenilearthritis; Juvenile diabetes; Kawasaki syndrome; Lambert-Eaton syndrome;Leukocytoclastic vasculitis; Lichen planus; Lichen sclerosus; Ligneousconjunctivitis; Linear IgA disease (LAD); Lupus (SLE); Lyme disease;Meniere's disease; Microscopic polyangiitis; Mixed connective tissuedisease; Mooren's ulcer; Mucha-Habermann disease; Multiple sclerosis;Myasthenia gravis; Myositis; Narcolepsy; Neutropenia; Ocular cicatricialpemphigoid; Osteoarthritis; Palindromic rheumatism; Paraneoplasticcerebellar degeneration; Paroxysmal nocturnal hemoglobinuria;Parsonnage-Turner syndrome; Pars planitis (peripheral uveitis);Pemphigus; Peripheral neuropathy; Perivenous encephalomyelitis;Pernicious anemia; POEMS syndrome; Polyarteritis nodosa; Type I, II, &III autoimmune polyglandular syndromes; Polymyalgia rheumatica;Polymyositis; Postmyocardial infarction syndrome; Postpericardiotomysyndrome; Progesterone dermatitis; Primary biliary cirrhosis; Psoriasis;Psoriatic arthritis; Idiopathic pulmonary fibrosis; Pyodermagangrenosum; Pure red cell aplasia; Raynauds phenomenon; Reflexsympathetic dystrophy; Reiter's syndrome; Relapsing polychondritis;Restless legs syndrome; Rheumatic fever; Rheumatoid arthritis;Sarcoidosis; Schmidt syndrome; Scleritis; Scleroderma; Sjogren'ssyndrome; Sperm & testicular autoimmunity; Stiff person syndrome;Subacute bacterial endocarditis; Sympathetic ophthalmia; Takayasu'sarteritis; Temporal arteritis/Giant cell arteritis; Thrombocytopenicpurpura; Autoimmune thyroid disease; Tolosa-Hunt syndrome; Transversemyelitis & necrotizing myelopathy; Ulcerative colitis; Undifferentiatedconnective tissue disease; Uveitis; Vasculitis; Vesiculobullousdermatosis; Vitiligo; and Wegener's granulomatosis. The more commonautoimmune diseases that are of especial interest include (a) connectivetissue diseases such as systemic lupus erythematosus, rheumatoidarthritis, systemic sclerosos (scleroderma), Sjogren's syndrome, (b)neuromuscular diseases such as multiple sclerosos, myasthenis gravis,Guillain-Barre syndrome, (c) endocrine diseases such as Hashimoto'sthryoiditis, Grave's disease, insulin-dependent (type 1) diabetes, and(d) gastrointestinal diseases such as inflammatory bowel disease(including Crohn's disease and ulcerative colitis), and (e) otherdiseases such as vasculitis syndromes, hematologic autoimmune diseases,and autoimmune skin diseases.

The autoimmune disease for example includes the presence ofautoantibodies. The autoantibody can bind specifically to host targetsor antigens, for example rheumatoid factor (e.g., in rheumatoidarthritis); topoisomerase (e.g., in scleroderma); myelin basic protein(e.g., in multiple sclerosis); basement membrane collagen type ivprotein (e.g., in Goodpasture's syndrome); ganglioside (e.g., inGuillain-Barré syndrome); platelets (e.g., chronic idiopathicthrombocytopenia); smooth muscle actin (e.g., in autoimmune hepatitis);bullous pemphigoid antigen 1 and 2; also called hemidesmosome antigens(e.g., in bullous pemphigoid); transglutaminase (e.g., in coeliacdisease); desmogein 3 (e.g., in pemphigus vulgaris); p62 or sp100 ormitochondrial (m2) antigens (e.g., in primary biliary cirrhosis);neutrophil cytoplasmic c-ANCA (e.g., in Wegener's granulomatosis);neutrophil perinuclear p-ANCA (e.g., Polyarteritis nodosa, Microscopicpolyangiitis, Churg-Strauss syndrome, Systemic vasculitides(non-specific)); double-stranded-DNA (e.g., in systemic lupuserythematosus); exosome complex (e.g., in Scleromyositis); Ro or Laantigen (e.g., in systemic lupus erythematosus and neonatal heart block,or primary Sjogren's syndrome); Smith antigen (e.g., in systemic lupuserythematosus); phospholipid antigen (e.g., in antiphospholipidsyndrome); SSA or SSB antigen (e.g., in Sjogren's syndrome); centromere(e.g., in CREST syndrome; mitochondria (e.g., in primary biliarycirrhosis); nicotinic acetylcholine receptor (e.g., in myastheniagravis); voltage-gated calcium channel (e.g., in Lambert-Eatonsyndrome); thyroid peroxidase (e.g., in Hashimoto's thyroiditis); TSHreceptor (e.g., in Graves' disease); Hu antigen (e.g., in paraneoplasticcerebellar syndrome); voltage-gated potassium channel (e.g., in limbicencephalitis and N-methyl-D-aspartate receptor (e.g., in encephalitis).More than one type of autoantibody can be associated with animmunological disorder or visa versa, and this list in not exhaustive.For example, autoantigens that have been identified in rheumatoidarthritis include joint-associated proteins such as collagen type II,human chondrocyte glycoprotein 39, and proteoglycans; as well as heatshock proteins, citrullinated filaggrin, immunoglobulin,glucose-6-phosphate isomerase, p205, and BiP.

The CD19-binding agent can be administered in an amount effective tomitigate at least one symptom of the autoimmune disorder. The inventionprovides treatment of an autoimmune disease that is refractory toconventional therapy with at least one CD19-binding agent. The CD19binding agent is optionally administered in combination with anothertherapy, e.g., surgery, anti-inflammatory drug therapy, hormone/enzymereplacement therapy, plasmapheresis and immunosuppressant therapy.Anti-inflammatory drug therapies include steroids, e.g., corticosteroidssuch as prednisone; as well as NSAIDs such as salicylates and other COXinhibitors. Hormone replacement therapy includes thyroid hormonereplacement (e.g., in Hashimoto's Thryoiditis). Immunosuppressant drugsinclude glucocorticoids, alkylating agents (e.g., cyclophosphamide,often effective in SLE), and antimetabolites (e.g., methotrexate,azathioprine and mercaptopurine). Other therapies include antithyroiddrug therapy or removal of the thyroid gland surgically or byradioiodine (e.g., in Grave's disease).

The CD19 binding agents, as well as ligand-drug conjugate compounds, arealso useful for treating or preventing a CD19-expressing cancer.Treatment or prevention of a CD19-expressing cancer, according to themethods described herein, is achieved by administering to a subject inneed of such treatment or prevention an effective amount of the CD19binding agent, or ligand-drug conjugate compound whereby the agent orcompound (i) binds to CD19-expressing cancer cells and (ii) exerts acytotoxic or cytostatic effect to, for example, deplete or inhibit theproliferation of the CD19-expressing cancer cells.

Cancers that can be treated or prevented by the methods described hereininclude, for example, B cell malignancies, including, for example, Bcell subtype non-Hodgkin's lymphoma (NHL) including low grade/follicularNHL, small lymphocytic (SL) NHL, intermediate grade/follicular NHL,intermediate grade diffuse NHL, diffuse large B-cell lymphoma,follicular lymphoma, high grade immunoblastic NHL, high gradelymphoblastic NHL, high grade small non-cleaved cell NHL and bulkydisease NHL; Burkitt's lymphoma; multiple myeloma; pre-B acutelymphoblastic leukemia and other malignancies that derive from early Bcell precursors; common acute lymphoblastic leukemia; chroniclymphocytic leukemia; hairy cell leukemia; Null-acute lymphoblasticleukemia; Waldenstrom's Macroglobulinemia; and pro-lymphocytic leukemia;light chain disease; plasmacytoma; osteosclerotic myeloma; plasma cellleukemia; monoclonal gammopathy of undetermined significance (MGUS);smoldering multiple myeloma (SMM); indolent multiple myeloma (IMM); orHodgkin's lymphoma, provided that the cancers express the CD19 antigen.

Therapeutic Uses of the Compounds of the Present Invention

In therapeutic applications, at least one CD19 binding agent (e.g., anantibody or an antibody-drug conjugate) is administered to a patientsuspected of, or already known to be suffering from, a CD19-associateddisorder, e.g., a cancer. The agent for example is administered in anamount sufficient to abolish, or at least lessen, at least one symptomof the disorder.

In prophylactic applications of treatment, at least one agent isadministered to a patient at risk of developing or suffering a relapseof a CD19-associated disorder. The patient is for example a patient inapparent remission of a CD19-associated disorder, for whom there is apossibility of a relapse, or a patient who is at enhanced risk of anincrease in at least one symptom of a CD19-associated disorder relativeto the general population. Patients known to be at high risk of aCD19-associated disorder or its relapse include, e.g., patientsdiagnosed with an aggressive form of the disorder, or with genetic orhistological abnormalities associated with the disorder or its staging(e.g., malignancy), or with an associated risk factor (e.g., familialhistory or another CD19 associated disorder or EBV infection).

The agent can be administered before a suspected onset or increase orexacerbation or relapse of the disorder, in an amount sufficient toeliminate, or reduce the risk of, or delay the onset or relapse of thedisorder.

The CD19 binding agents and ligand-drug conjugate compounds are usefulfor treating cancer and other diseases in which CD19 is expressed oroverexpressed, relative to normal (e.g., non-cancerous tissue). The CD19binding agents can also be used to treat CD19-associated disorders inwhich CD19 is not overexpressed relative to normal. For example, thedisorder can include an increased count of CD19-positive B cells. Insome embodiments, the CD19 binding agents and ligand-drug conjugatecompounds are administered alone. In other embodiments, the CD19 bindingagents and the ligand-drug conjugate compounds are coadministered withanother therapeutic agent, or administered sequentially. In someembodiments, the CD19 binding agents and ligand-drug conjugate compoundscoadministered with standard of care chemotherapeutics, or administeredsequentially.

The response of the patient can be monitored by determining the effectof the agent on a CD19-associated disorder.

Treatment of Cancer

The CD19 binding agents and ligand-drug conjugate compounds are usefulfor inhibiting the multiplication of a tumor cell or cancer cell,causing apoptosis in a tumor or cancer cell, or for treating cancer in apatient. The compounds can be used accordingly in a variety of settingsfor the treatment of cancers. The conjugate compounds provideantigen-specific tumor or cancer targeting, thus reducing potentialtoxicity of the Drug.

In some embodiments, the cancer (e.g., B cell lymphoma and a leukemia)is a cancer expressing or over-expressing CD19 (i.e., relative to normaltissue), such as in B-lymphocytes.

Multi-Modality Therapy for Cancer

Cancers, including, but not limited to, a tumor, metastasis, or otherdisease or disorder characterized by uncontrolled cell growth, can betreated or prevented by administration of compounds according to thepresent invention, i.e., CD19 binding agents or ligand-drug conjugatecompounds.

In some embodiments, methods for treating or preventing cancer areprovided, including administering to a patient in need thereof aneffective amount of a compound of the present invention and achemotherapeutic agent. In one embodiment, the chemotherapeutic agent isthat with which treatment of the cancer has not been found to berefractory. In another embodiment, the chemotherapeutic agent is thatwith which the treatment of cancer has been found to be refractory. Thecompounds of the present invention can be administered to a patient thathas also undergone surgery as treatment for the cancer.

In one embodiment, the additional treatment is radiation therapy.

In a specific embodiment, the CD19 binding agent or CD19 ligand-drugconjugate compound is administered concurrently or sequentially with thechemotherapeutic agent and/or with radiation therapy. In anotherspecific embodiment, the chemotherapeutic agent or radiation therapy isadministered prior or subsequent to administration of the CD19 bindingagent, or anti-CD19 ligand-drug conjugate compound. In some embodiments,the chemotherapeutic agent or radiation therapy is administered at leastan hour, five hours, 12 hours, a day, a week, a month, several months(e.g., up to three months), prior or subsequent to administration of acompound of the present invention.

Where a compound of the present invention and chemotherapeutic drug(s)are administered separately, the number of dosages of each compoundgiven per day, may not necessarily be the same, e.g. where one compoundmay have a greater duration of activity, and will therefore, beadministered less frequently. The compound of the present invention andthe chemotherapeutic drug(s) can be administered via the same ordifferent routes of administration. They can be administered accordingto simultaneous or alternating regimens, at the same or different timesduring the course of the therapy, concurrently in divided or singleforms. Administration of either or both agents can be on a continousbasis, e.g., by infusion or via an implanted reservoir.

A chemotherapeutic agent can be administered over a series of sessions.Any one or a combination of the following chemotherapeutic agents can beadministered (see infra). With respect to radiation, any radiationtherapy protocol can be used depending upon the type of cancer to betreated. For example, but not by way of limitation, x-ray radiation canbe administered; in particular, high-energy megavoltage (radiation ofgreater that 1 MeV energy) can be used for deep tumors, and electronbeam and orthovoltage x-ray radiation can be used. Gamma-ray emittingradioisotopes, such as radioactive isotopes of radium, cobalt and otherelements, can also be administered.

Additionally, methods of treatment of cancer with a compound of thepresent invention are provided as an alternative to chemotherapy orradiation therapy where the chemotherapy or the radiation therapy hasproven or can prove too toxic, e.g., results in unacceptable orunbearable side effects, for the subject being treated. The subjectbeing treated can, optionally, be treated with another cancer treatmentsuch as surgery, radiation therapy or chemotherapy, depending on whichtreatment is found to be acceptable or bearable.

Compounds of the present invention can also be used in an in vitro or exvivo fashion, such as for the treatment of certain cancers.

Multi-Drug Therapy for Cancer

Methods for treating cancer including administering to a patient in needthereof an effective amount of an CD19 binding agent or ligand-drugconjugate compound and another therapeutic agent that is an anti-canceragent.

In some embodiments, the other therapeutic agent will be an agent thatis standard of care for the specific disease to be treated or is part ofa salvage regimen for the specific disease to be treated. Anti-canceragents and chemotherapeutic regimens include, for example, anti-cancerantibodies, including, for example, anti-CD52 antibodies (e.g.,Alemtuzumab), anti-CD20 antibodies (e.g., Rituximab), and anti-CD40antibodies (e.g., SGN40); chemotherapeutic regimens including, forexample, CHOP (cyclophosphamide, doxorubicin, vincristine, andprednisone); CVP (cyclophosphamide, vincristine, and prednisone); RCVP(Rituximab+CVP); RCHOP (Rituximab+CHOP); RICE (Rituximab+ifosamide,carboplatin, etoposide); RDHAP, (Rituximab+dexamethasone, cytarabine,cisplatin); RESHAP (Rituximab+etoposide, methylprednisolone, cytarabine,cisplatin); gemcitabine; combination treatment with vincristine,prednisone, and anthracycline, with or without asparaginase; combinationtreatment with daunorubicin, vincristine, prednisone, and asparaginase;combination treatment with teniposide and Ara-C (cytarabine);combination treatment with methotrexate and leucovorin; combinationtreatment with bleomycin, doxorubicin, etoposide, mechlorethamine,prednisone, vinblastine, and vincristine; small molecule inhibitors; andproteosome inhibitors including, for example, bortezomib.

The present invention encompasses methods of treating lymphomas usingthe described CD19 binding agents or ligand-drug conjugate compoundsalone or in combination therapy with, for example, anti-lymphomaantibodies, including, for example, anti-CD20 antibodies, i.e.,Rituximab, and/or anti-CD40 antibodies, i.e., SGN-40.

The present invention encompasses methods of treating lymphomas usingthe described CD19 binding agents or ligand-drug conjugate compoundsalone or in combination therapy with, for example, chemotherapeuticregimens for the treatment of lymphomas including, for example, CHOP(cyclophosphamide, doxorubicin, vincristine, and prednisone), CVP(cyclophosphamide, vincristine, and prednisone) and/or otheranthracycline B chemotherapy regimens.

The present invention encompasses methods of treating indolent lymphomasusing the described CD19 binding agents or ligand-drug conjugatecompounds alone or in combination therapy with, for example, RCVP(Rituximab+CVP) and/or RCHOP (Rituximab+CHOP).

The present invention encompasses methods of treating subjects sufferingfrom relapsed or refractory lymphoma using the described CD19 bindingagents or ligand-drug conjugate compounds alone or in combinationtherapy with, for example, RICE (Rituximab+ifosamide, carboplatin,etoposide), RDHAP, (Rituximab+dexamethasone, cytarabine, cisplatin),RESHAP (Rituximab+etoposide, methylprednisolone, cytarabine, cisplatin),gemcitabine and/or an immune modulatory drugs, i.e., lenalidomide.

The present invention encompasses methods of treating a subject that hasrelapsed disease or that is refractory to treatment with Rituximab orother therapy for the treatment of cancer, e.g., CHOP, CVP, CHOP, RICE,RDHAP, RCHOP, RCVP, RESHAP. In one aspect, the methods include, forexample, administering a ligand-drug conjugate of the present inventionto the subject. In certain embodiments, the ligand-drug conjugatecomprises a CD19 binding agent conjugated to an auristatin compound. Inone aspect, the CD19 binding agent is a humanized BU12 antibody.

The present invention encompasses methods of treating a subject that hasa cancer characterized by the level of CD21 expression. The cancer canhave no, low levels, or high levels of CD21 expression. In one aspect,the methods include, for example, administering a ligand-drug conjugateof the present invention to the subject. In certain embodiments, theligand-drug conjugate comprises a CD19 binding agent conjugated to anauristatin compound. In one aspect, the CD19 binding agent is ahumanized BU12 antibody.

The present invention encompasses methods of treating ALL using thedescribed CD19 binding agents or ligand-drug conjugate compounds aloneor in combination therapy with, for example, a chemotherapeutic regimenthat includes the combination of vincristine, prednisone, andanthracycline, with or without asparaginase. Alternativechemotherapeutic regimens include, for example, combinations ofdaunorubicin, vincristine, prednisone, and asparaginase; combinations ofteniposide and ara-C(cytarabine); combinations of methotrexate andleucovorin; combinations of bleomycin, doxorubicin, etoposide,mechlorethamine, prednisone, vinblastine and vincristine (“Stanford 5Regimen”).

Also within the scope of the invention are kits comprising an isolatedCD19 binding agent that specifically binds to human CD19 or aligand-drug conjugate comprising a CD19 binding agent, and instructionsfor use. The kit can further contain a least one additional reagent.Kits typically include a label indicating the intended use of thecontents of the kit. The term label includes any writing, or recordedmaterial supplied on or with the kit, or which otherwise accompanies thekit.

The invention also includes diagnostic use of a humanized CD19 antibody.For example, the humanized CD19 antibody can be used as a diagnosticimaging agent alone and/or in combination with other diagnostic imagingagents and/or in conjunction with therapeutic applications. Thediagnostic agent can be used in vivo in human patients known to have orhave had a CD19-associated disorder. Optionally, the disorder includesCD19-positive cells that are discrete localized, e.g., in a solid tumor.

In one such method, the CD19 binding agent can be directly or indirectlylabeled with a detectable label, such as a fluorophore, and optionallycontacted with a target cell or a patient sample in vitro or in vivo.The presence and/or density of CD 19 in a sample or individual can bedetermined. In vivo methods of determination can include imagingtechniques such as PET (positron emission tomography) or SPECT (singlephoton emission computed tomography).

The patient or sample (an optionally a control as well) is for examplecontacted with a CD19 binding agent under conditions that allow forformation of a complex between the agent and CD19 antigen if present.Complex formation is then detected in the test patient and compared tobinding in a control (e g, using an FACS analysis or Western blotting).Any statistically significant difference in the formation of complexesbetween the control and the test sample/patient is indicative of thepresence of a CD19-associated disorder. The control can be e.g., asimilar reading taken from the same patient at a different location ortimepoint or a reading taken from non-diseased subjects, or apredetermined statistical value based on multiple readings taken fromindividuals selected at random or not known to be suffering from thedisorder. The diagnostic tests can be used to identify patients with aCD19-associated disorder, or to determine the extent of such a disorderin a particular patient, or to monitor the course of a disorder overtime, or the effect of a chosen treatment on a disorder.

All publications and patent documents cited above are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

The following examples of specific aspects for carrying out the presentinvention are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way.

EXAMPLES Example 1 Design of Humanized BU12 Heavy Chain Variable Region:

BU12 V_(H) was aligned to functional human germline V_(H) exons.Selection of the V_(H) exon was made based on framework homology andcanonical structure. Human germline V_(H) exons V_(H)2-70 and V_(H)4-31were selected to provide frameworks for humanization. Human germlineJ_(H)4 exon was selected to provide humanized FR4 sequence based on itsidentity (85%) with FR4 of BU12 V_(H).

BU12 V_(H) was aligned against the mouse V_(H) exon germline sequencesto identify regions of somatic mutation which may have structuralimplications. BU12 V_(H) was found to have high homology to thefunctional CB17H-10 V_(H) exon with potential framework regions ofsomatic mutation at H75, H82A and H89.

BU12 V_(H) was aligned against the selected human germline V_(H) exon(V_(H)2-70 or V_(H)4-31) and differences between BU12 V_(H) and thehuman framework at residues described in the literature to effect CDRstructure or V_(H)/V_(L) interactions were identified. For the V_(H)4-31such residues changes were found at positions H24, H27, H29 and H71.Additionally non-homologous framework regions were identified and thecrystal structure of a homologous V_(H) domain (1ETZ) was used todetermine the positions of non-homologous residues and assess theirlikely impact on CDR structure.

Humanizing Mutations in Heavy Chain Variants

V_(H) V_(H) Exon Donor Variant Acceptor Sequence Framework ResiduesV_(H)A VH2-70 None V_(H)B VH2-70 H75 V_(H)C VH2-70 H79 V_(H)D VH2-70H81, H82, H82A, H82B, H82C V_(H)E VH2-70 H89 V_(H)F VH4-31 None V_(H)GVH4-31 H71 V_(H)H VH4-31 H24, H27, H29 V_(H)I VH4-31 H24, H27, H29, H71V_(H)J VH4-31 H75 V_(H)K VH4-31 H78, H79 V_(H)L VH4-31 H89

Non-homologous FR Residues BU12 V_(H) vs. V_(H)2-70 Position ChangeComments H41 S → P Loop region—exclude H75 S → K Possible somaticmutation/ charge change H79 F → P Aromatic in core H81 K → T Core(continuous region) H82 I → M Core (continuous region) H82A A → T Core(continuous region) H82B S → N Core (continuous region) H82C V → M Core(continuous region) H84 T → P Loop region—exclude H89 A → T Possiblesomatic mutation

Specific Mutations in BU12 Heavy Chain Variants

Variant H75 H79 H81 H82 H82A H82B H82C H89 cBU12 VH S* F* K* I* A* S* V*A* VH2-70 K V T M T N M T HA K V T M T N M T HB S* V T M T N M T HC K F*T M T N M T HD K V K* I* A* S* V* T HE K V T M T N M A* *Mouse residues

Non-homologous FR Residues BU12 VH vs. VH4-31 Position Change CommentsH3 T → Q Surface accessible distant from CDRs—exclude H24 F → V ImpactsCDR1 structure H27 F → G Impacts CDR1 structure H29 L → I Impacts CDR1structure H41 S → P Loop region—exclude H71 K → V Impacts CDR2 structureH75 S → K Possible somatic mutation/charge change H78 V → F Aromatic incore H79 F → P Aromatic in core H83 D → T Loop region—exclude H89 A → VPossible somatic mutation

Specific Mutations in BU12 Heavy Chain Variants

Variant H24 H27 H29 H71 H75 H78 H79 H89 cBU12 VH* F* F* L* K* S* V* F*A* VH4-31 V G I V K F S V HF V G I V K F S V HG V G I K* K F S V HH F*F* L* V K F S V HI F* F* L* K* K F S V HJ V G I V S* F S V HK V G I V KV* F* V HL V G I V K F S A* *Mouse residues

Example 2 Design of Humanized BU12 Light Chain Variable Region:

BU12 V_(L) was aligned to functional human germline V_(H) exons. Usageof L6 is high (˜11%) so this was chosen as the best framework for BU12V_(L) humanization. A10, with the best homology to BU12 V_(L) was alsochosen. Human germline J_(κ)2 exon was selected to provide humanized FR4sequence based on its identity (77%) to FR4 of BU12 V_(L).

BU12 V_(L) was aligned against the mouse V_(L) exon germline sequencesto identify regions of somatic mutation which may have structuralimplications. The closest matches were ac4, kn4 and kk4. Potential sitesof somatic mutation were identified at positions L40, L41, L42, L69,L71, L72 and L83.

BU12 V_(L) was aligned against the selected human germline and V_(L)exon (L6 or A10) and differences between BU12 V_(L) and the humanframework at residues described in the literature to effect CDRstructure or V_(H)/V_(L) interactions were identified. Such residuedifferences occur at positions L2 and L71. Additionally non-homologousframework regions were identified and the crystal structure of ahomologous V_(L) domain (1QOK) was used to determine the positions ofnon-homologous residues and assess their likely impact on CDR structure.

Humanizing Mutations in Light Chain Variants

V_(L) V_(H) Exon Donor Variant Acceptor Sequence Framework ResiduesV_(L)A VL-L6 None V_(L)B VL-L6 L2 V_(L)C VL-L6 L71 V_(L)D VL-L6 L2, L71V_(L)E VL-L6 L40, L41, L42 V_(L)F VL-L6 L69, L70, L71, L72 V_(L)G VL-L6L83 V_(L)H VL A10 None V_(L)I VL A10 L2, L71

Non-homologous FR Residues BU12 VL vs. L6 and A10 Position ChangeComments L2 N → I L2 known to impact CDR1 structure L40 S → P Possiblesomatic mutation L41 S → G Possible somatic mutation L42 T → Q Possiblesomatic mutation L69 N → T Possible somatic mutation L70 S → D Charge instrand packing against CDR1 L71 H → F Somatic mutation/L71 known toimpact CDR1 structure L72 F → T Possible somatic mutation L83 V → FPossible somatic mutation

Specific mutations in BU12 1ight chain variants: Variant L2 L40 L41 L42L69 L70 L71 L72 L83 cBU12 VL* N* S* S* T* N* S* H* F* V* L6 I P G Q T DF T F LA I P G Q T D F T F LB N* P G Q T D F T F LC I P G Q T D H* T FLD N* P G Q T D H* T F LE I S* S* T* T D F T F LF I P G Q N* S* H* F* FLG I P G Q T D F T V* *Mouse residues

Specific Mutations in BU12 Light Chain Variants

Variant L2 L71 cBU12* N* H* A10 I F LH I F LI N* H* *Mouse residues

Example 3

A panel of anti-CD19 antibodies was screened on a panel of CD19+ NHLcell lines (FIG. 15). All of the antibodies evaluated were able todeliver drug although there were differences between the cell lines.

IC₅₀ (ng/mL) of various anti-CD19 Antibodies CD19 linked with 2°-ADCCell Line Disease Type Molecules/Cell LT19 HIB19 cBU12 SJ25-C1 B-C3 CA46Burkitt's Lymphoma, 60527 4 1 7 4 17 EBV− HS Sultan Burkitt's Lymphoma,59669 112 97 100 150 234 EBV+ HT Diffuse Mixed 35813 111 ~1000 238 ND NDLymphoma MC 116 Undifferentiated 29210 192 188 186 ~200 195 LymphomaRamos Burkitt's Lymphoma, 34377 1 1 5 3 13 EBV− Toledo Diffuse LargeCell 28657 584 ~1000 ~1000 512 359 Lymphoma

Anti-CD19 antibodies deliver 2°-goat-anti-mouse-vcMMAF to CD19⁺ celllines. Cell lines were cultured with different anti-CD19 antibodiescross-linked with a 2-fold excess of goat-anti-mouse ADC(187.1-vcMMAF8). Cultures were incubated for 96 hours and labeled with50 μM resazurin. There was no effect of 187.1-vcMMAF on the growth ofany of the cell lines tested. Values are the mean±SD of four replicateswithin a single experiment.

Example 4

Antitumor activity of anti-CD19 antibody-drug conjugate compounds onRamos tumor model in SCID mice was determined. The results generallyshow that murine and chimeric BU12 antibody drug conjugates had pooractivity as compared to other chimeric anti-CD19 antibody drugconjugates and as compared to humanized BU12 antibody drug conjugates.See FIGS. 3, 4, 5, 7 and 8.

Example 5

Variants of humanized BU12 antibody, in which amino acid residues in theFc domain of IgG₁ known to be important for binding to FcγR can bemutated to impair binding to one or more FcγR, can be generated usingstandard molecular biology techniques.

For example, IgG1v1 contains the following mutations: E233P:L234V:L235A,according to the Kabat numbering scheme. The amino acid sequence ofIgG1V1 is shown in SEQ ID NO:35.

Further Fc domain variants of humanized anti-CD19 antibodies can besimilarly generated, including, for example, Fc domain variants with oneor more non-conservative amino acid substitutions, introduction of oneor more cysteine residues, or introduction of one or more sites forN-linked glycosylation, in or in proximity to the Fc domain involved inthe binding interaction to one or more Fcγ receptors.

Example 6

Preparation of a hBU12 Antibody Drug Conjugate

One hundred thirty milligrams of the hBU12 mAb (Lot #'s PR208 (69 mg)and 1033154 (100 mg)) were combined and concentrated to provide 141 mgat a concentration of 10.8 mg/mL, based on a molecular weight of 150 kDand an extinction coefficient of 1.47 AU·mL·mg⁻¹·cm⁻¹.

The auristatins MMAE and MMAF were conjugated to the purified antibodyas follows. The antibody (130 mg, 867 nmol) was incubated 45 min at 37°C. with 2.17 nmol of TCEP (representing a 25% excess of reductant forthe desired reduction level of 4 free thiols per antibody) with 1 mMDTPA as a cation scavenger. The reduction level was determined byperforming a microscale test conjugation with the following testcompound:

The drug loading distribution was characterized by HIC chromatography.This mAb exhibited a reduction pattern occasionally seen with murineantibodies, wherein the distribution is weighted at 0 and 10 drugs perantibody, with 4- and 6-loaded antibody being represented at lowerlevels. The mean drug loading was higher than desired: 4.9 drugs/Ab.Incremental quantities of DTNB (217 nmol, then 303.8 nmol) were added tore-oxidize antibody disulfides, thereby reducing the drug loading levelto an assayed level of 4.1 drugs/Ab.

The partially reduced mAb (97 mg, 647 nmol) was conjugated with MMAF byaddition of 795 μL of DMSO to the approximately 9.0 mL of mAb solution,followed by 203.1 μL of a 19.1 mM DMSO solution of the followingcompound, maleimidocaproyl-Val-Cit-MMAF (3.88 μmol).

The conjugation reaction was allowed to proceed for 100 minutes at 0° C.Residual maleimidocaproyl-Val-Cit-MMAF was quenched by addition of 194μL of 100 mM N-acetyl cysteine. The reaction mixture was then dialyzedagainst 4 L of PBS three times using a 25000 MWCO membrane at 4° C. toremove DMSO, unreacted or quenched drug, and other small-moleculecontaminants resulting from the conjugation process, and concentrated.The product contained 4.1 drugs/Ab

Example 7

Activity of the Anti-CD19 Auristatin Antibody Drug ConjugatehBU12-vcMMAE Against Rituximab Sensitive and Resistant Lymphomas and inCD21 High and Low Lymphomas:

Materials and Methods

Flow Cytometric Analysis to Determine CD19 and CD21 Expression Levels onTumor Cell Lines:

To evaluate CD19 and CD21 copy numbers on tumor cell lines, cells wereincubated for 30 minutes on ice with PE-conjugated murine anti-CD19 andanti-CD21 antibodies (BD Pharmingen, San Diego, Calif.), washed withcold staining medium and evaluated with a Becton Dickison FACScan flowcytometer. Quantitative determination of CD19 and CD21 on the cellsurface was determined using a DAKO QiFiKit flow cytometric indirectimmunofluorescence assay and murine antibodies as described by themanufacturer (DAKO A/S, Glostrup, Denmark).

Saturation Binding Studies to Determine Binding Affinity:

Cells were incubated with 10 μg/ml hBU12 or hBU12-vcMMAE for 0.5 h at 4C, and washed. One set of cells was transferred to 37° C. and harvestedat selected timepoints. For detection, a secondary PE-conjugatedantibody was used and the amount of remaining surface-bound antibodydetermined by flow cytometry. Alternatively, cells were incubated on icewith AlexaFluor488-labeled hBU12 antibody or drug conjugates for 1 h,washed with cold PBS, and binding assessed with a Becton DickisonFACScan flow cytometer. The apparent Kd values were determined using theOne Site Binding algorithm from Prism (GraphPad Software, San Diego,Calif.).

CD19 Internalization Kinetic Studies:

To generate radiolabeled antibody-drug conjugates, custom synthesized[3H]-vcMMAE (24.7 Ci/mmol, Moravek Biochemicals, Brea, Calif.) was usedto prepare the radiolabeled hBU12-vcMMAE conjugate. Calculations ofradioactivity were made. The amount of free drug found inside of thecells from 1 mL of culture was added to the amount of free drug detectedin 1 mL of culture medium and this value was used to determine theconcentration of total drug released in the cell culture. Triplicateresults were averaged and the standard deviation for those values wascalculated using the STDEVPA function in Microsoft Excel.

Lysosomal Co-Localization Studies of hBU12 and hBU12-ADCs:

Ramos cells were incubated with 1 ug/ml hBU12 or hBU12-ADCs on ice orfor 20 minutes or 4 hours at 37° C. After the incubation, the cells werewashed with cold PBS to remove unbound antibody or ADC and then fixedand permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose,Calif.). The antibody and ADCs were detected with AlexaFluor-488 labeledgoat anti-human IgG (Molecular Probes, Eugene, Oreg.). Lysosomalcompartments were visualized by staining with AlexaFluor647-labeledLAMP-1 antibody (mouse CD107, BD Biosciences). Nuclear compartments werestained with DAPI (4′, 6-diamidino-2-phenylindole, Roche, Basel,Switzerland). Fluorescence images were acquired with a Carl ZeissAxiovert 200M microscope.

Cytotoxicity and Growth Arrest Assays:

Tumor cells were incubated with hBU12 and the drug conjugates for 96 h.Cell viability was measured by Alamar Blue (Biosource International,Camarillo, Calif.) dye reduction as previously reported (Doronina, 2003#1834). Cells were incubated for 4 h with the dye and dye reductionmeasured on a Fusion HT fluorescent plate reader (Perkin Elmer, Waltham,Mass.). Results are reported as IC50, the concentration of compoundneeded to yield a 50% reduction in viability compared to vehicle-treatedcells (control=100%). For growth arrest and apoptosis studies, cellswere first treated with the antibody and ADCs and then processed usingthe Annexin V-FITC Apoptosis Detection kit (BD Pharmingen), according tothe manufacturer's directions. For analysis of cell cycle positionfollowing exposure to ADCs, the cells were labeled for 20 minutes withbromodeoxyuridine (BrdUrd, Sigma, St. Louis, Mo.). Nascent DNA synthesiswas detected using an anti-BrdUrd antibody (BD Biosciences) and totalDNA content was detected with propidium iodide (PI). Cells were thenanalyzed by flow cytometry.

In Vivo Model of Subcutaneous Lymphomas and Disseminated HumanLeukemias:

Localized, subcutaneous and disseminated models of B cell lymphomas wereestablished in SCID mice. For the subcutaneous model, 5×10⁶ lymphomacells were implanted into the right flank of female mice. hBU12 and-ADCs or a control compound were administered when tumor volumes reached100 mm³. Tumor size was monitored at least twice weekly. To establishdisseminated disease, 1×10⁵ Nalm6 cells or RS4; 11 cells in 0.2 ml PBSwere injected into the lateral tail vein of C.B.-17 SCID mice (Harlan,Indianapolis, Ind.). Mice were treated with test compounds 7 days aftercells injection and monitored at least twice per week. Mice wereterminated when they exhibited signs of disease including weight loss of15-20%, hunched posture and lack of grooming, cranial swelling and hindlimb paralysis. Treatment schedules are as indicated in the figurelegends.

Statistical Analysis:

Tumor quadrupling or triplication times (as indicated) were chosen astime to endpoint (TTE), which were determined by using a non-linearregression analysis for exponential growth of each individual tumorgrowth data set from each experimental animal. The tumor quadruplingtime was calculated based on the tumor volume at the beginning oftreatment. Animals that did not reach the endpoint were assigned a TTEvalue equal to the last day of the study. % TGD (tumor growth delay)reflects the delay in reaching TTE relative to control treated tumors,which was determined by using the formula: % TGD=[(T−C)/C]×100, where Tand C are the median times in days for treated and control groups, toreach TTE, using the start of treatment as day 1. Statistical analysisand graphic presentations were conducted using Graphpad Prism Softwareversion 4.01 (Graphpad, San Diego, Calif.). Median tumor growth curvesshow group median tumor volumes as a function of time. The Log rank testwas used to analyze the significance of the differences between TTE oftreated and control tumor groups, with differences deemed significant(*) at 0.01<P<0.05, and highly significant (**) at P<0.01. In a CRresponse, the tumor volume is less than 13.5 mm³ for three consecutivemeasurements during the course of the study. A durable response (DR) isdefined as complete absence of palpable tumor during the entireexperiment. Standard Pearson correlation analysis (two tailed) wasemployed, using a 95% confidence interval, to determine significantcorrelations between CD19 and CD21 expression levels and in vitrocytotoxicity.

Development of Rituxan Resistant Ramos and Raji Tumors:

Parental cells were implanted into 40 SCID mice at a concentration of5×10⁶ cells per mouse. 2 days following cell implant, mice were treatedwith rituximab at 8 mg/kg every other day for a total of 9 doses. Out ofthe 40 mice, roughly 6 developed tumors, when the tumors wereapproximately 300-400 mm3, the mice were euthanized and the tumors werecollected aseptically. Tumors were made into a single cell suspensionthrough disassociation through a nylon filter. While in culture thecells were continuously exposed to various levels of rituximab up to 100ug/ml. Cell viability was verified several times per week. After oneweek in culture the cells were implanted into 30 SCID mice. Two daysafter implant the mice were treated with rituximab at 12 mg/kg in theschedule as before. The in vitro and in vivo selection was repeated oncemore in 10 SCID mice. The resulting tumors were processed into singlecell suspension and frozen in liquid nitrogen. The Raji R2 and Raji H4cell lines were generated as described in Czuczamn et al., Clin CancerRes. 2008; 14:1561-1570.

Pharmacokinetic Characteristics of hBU12-vcMMAE(4) Conjugates:

Single doses of hBU12-vcMMAE were administered intra-peritoneally tonaïve SCID mice. The serum samples were collected at scheduled intervalsover a period of 11 weeks to obtain composite pharmacokinetic profiles.The samples were analyzed for antibody drug conjugate concentrations bya qualified multiplex bead-capture assay using an anti-MMAE antibody.The pharmacokinetic analysis was done using non-compartmental andcompartmental methods.

Results

Lack of Correlation Between CD19 and CD21 Expression and Potency ofhBU12-vcMMAE Against ALL, CLL and NHL Tumor Cell Lines Grown in Culture:

In order to determine the potency of hBU12-vcMMAE, CD19 positivelymphoma and leukemia cells representing Burkitt's lymphoma, diffuselarge B-cell lymphoma (DLBCL), follicular lymphomas (FL), and acutelymphocytic leukemia (ALL) were exposed to increasing concentrations ofhBU12-vcMMAE. In addition, the cell surface copy numbers of CD19 andCD21 were determined in order to study a potential correlation betweenexpression of these genes with anti-tumor activity. Potent cytotoxicactivity of the hBU12-vcMMAE conjugate was noticed in 15 out of 17 CD19positive tumor cell lines tested. The T-cell lymphoma cell line Jurkatwas used as CD19 negative control cell line. The absence of activity ofhBU12-vcMMAE on these control cells suggested that the anti-tumoractivity was target dependent. A lack of significant correlationsbetween ADC potency and the levels of CD19 (p=0.45, R2=0.038) and CD21(p=0.55, R2=0.028) expression was noticed (data not shown). In addition,similar potent cytotoxic effects of hBU12-vcMMAE against ALL cell lineswere found. In sum, the data demonstrates that CD19 and CD21 expressionlevels and the tumor subtypes are insufficient to predict thesensitivities of lymphoma and leukemia cell lines towards auristatinbased ADCs.

Internalization Kinetics and Intracellular Trafficking of hBU12-vcMMAEin NHL Cell Lines:

A critical parameter previously shown to determine the anti-tumoreffects of certain auristatin based ADCs is the ability of the targetantigen to internalize and to translocate to the lysosomal compartmentfollowing ligation by the antibody. To study these processes, CD21 low(Ramos and SUDHL-4) and CD21 high tumor cell lines (Raji, Daudi) wereincubated with hBU12-vcMMAE and the internalization kinetics byfluorescence activated cell sorting (FACS) was determined. As shown inFIG. 15, hBU12 and hBU12-vcMMAE conjugates internalized rapidly in CD21low Ramos cells, and >50% of the compounds internalized within 60minutes post incubation. Somewhat slower internalization kinetics ofhBU12-vcMMAE were found in CD2 1high Raji and Daudi cells. However, thesmall differences in internalization kinetics did not significantlyaffect potency, and comparable IC50 values between CD21 low Ramos cellsand CD21 high lymphomas were found. Combined, the findings demonstratethat the internalization kinetics of hBU12-vcMMAE on different tumorcell lines does not correlate with cytotoxicity in vitro. Intracellulartrafficking of hBU12 and hBU12-vcMMAE conjugates in NHL cell lines wasinvestigated. For this purpose, CD21low Ramos and SUDHL4 cells wereincubated with either naked antibody or hBU12-vcMMAE.Co-immunofluorescence studies revealed that the majority of internalizedhBU12 localized to lysosomes, starting as early as 15 minutes postincubation. Comparable subcellular translocation of hBU12 and conjugatesto the lysosomal compartment was observed between CD21 low Ramos or HTand CD21 high Daudi and Raji cells (data not shown). Combined, thefindings demonstrate that internalization kinetics alone areinsufficient to explain the differences in hBU12-vcMMAE potenciesagainst different NHL cell lines.

Free Drug Release by hBU12-vcE in Rituximab Sensitive and Resistant,CD21 High and Low Lymphoma Cell Lines:

MMAE interferes with microtubule stability in the cytoplasmiccompartment and thus, the amounts of active, free MMAE drug released intumor cells is critical for anti-lymphoma effects. To investigate thesesaspects, CD21 high Daudi and CD21 high, rituximab resistant Raji R2 andRaji 4H cells were incubated with hBU12-vcMMAE and the levels of freedrug released with CD21 low Ramos cells were compared. The cellularrelease of free, active drug was quantified by combining theradioactivity that was retained within cells and that had escaped intothe supernatant over time. There was no difference in free drug releasebetween CD21 low cells (Ramos) and CD21 high cells (Daudi, Table 2).Therefore, it is unlikely that variations in free drug release accountfor the >50 fold differences in the IC50 values between these differentlymphoma cell lines. In conclusion, high CD21 levels may only minimallyinterfere with the intracellular release of free drug from internalizedhBU12-vcMMAE.

Efficacy of hBU12 conjugates in models of NHL and ALL hBU12-vcMMAE wastested in single dose and multi dose experiments using different NHLcell lines xenografted into SCID mice (FIG. 16A-E and data not shown).When tested against Burkitt's lymphoma, 7/8 durable responses wereobserved at the 3 mg/kg hBU12-vcMMAE dose, administered q4dx4 (FIG.16A)). When tested against DOHH2 tumor (Follicular lymphoma),significant inhibition of tumor growth rates as illustrated by 2/10 DRsat the 3 mg/kg dose level (FIG. 16B) were observed. When tested againstSUDHL4 lymphomas (DLBCL), there was also a significant inhibition oftumor growth rates (FIG. 16C). In the disseminated RS4; 11 modelrepresenting ALL, a significant increase in survival of mice treatedwith hBU12-vcMMAE was observed, resulting in a delay of disease onsetfrom ˜45 days in control or untreated animals to >90 days in micetreated with 3 mg/kg of vcE conjugates (FIG. 16D). Similar observationswere made when testing hBU12-vcMMAE in a second model of disseminatedALL (Nalm6), where single dose administration resulted in 30-60% durableresponses FIG. 16E). Combined, these data demonstrate potent anti-tumoreffects of hBU12-vcMMAE in different models of NHL and ALL, irrespectiveof their CD21 expression status.

Antitumor Activity hBU12-vcE Against Rituximab Resistant Lymphomas:

In order to develop a preclinical models that mimick the refractorinessof NHL tumors to rituximab treatment, Ramos tumor cells were generatedthat were rendered refractory by repeated in vitro and in vivo passagingof tumor cells in mice, concurrent with rituximab treatment as describedin materials and methods. To determine the expression levels of CD20 andCD19, cells from R-Ramos tumors were isolated and comparable levels ofCD20 and CD19 expression in rituximab resistant Ramos tumors were found(FIG. 17C). Compared with high doses of rituximab (12 mg/kg, q4dx4),hBU12-vcMMAE treatment resulted in a significant difference in % tumorgrowth delay (TGD) between the parental and rituximab resistant tumors.Importantly, similar antitumor activities by hBU12-vcMMAE in rituximabsensitive and resistant cell lines were found, suggesting that themechanism rendering NHL cells resistant to rituximab does interfere withhBU12-vcMMAE potency. To further validate these findings, hBU12-vcMMAEwas tested in conjunction with two additional, rituximab resistant NHLcell lines described previously (Raji 2R and 4RH 27; FIG. 17D and datanot shown), and both cell lines were shown to express similar levels ofCD19 and CD20 compared to the parental clone. In support of the previousfindings testing rituximab resistant Ramos tumors, hBU12-vcMMAEtreatment induced similar durable response compared to the rituximabsensitive parental Raji clone. In conclusion, potent anti-tumoractivities for CD19-ADCs on rituximab resistant cell lines wereobserved.

Example 8

Role of Effector Cells in Mediating Therapeutic Effects of the HumanizedAnti-CD19 Antibody hBU12 in Preclinical Models of NHL and ALL:

The ability of the humanized anti-CD19 antibody hBU12 to induce CDC,ADCC and ADCP against human lymphoma and leukemia cell lines wasinvestigated. Potent ADCC and ADCP was found and ADCC was slightlyreduced when hBU12 was conjugated to the vcMMAE drug linker. When testedon activated, human primary B-cell isolates, direct anti-proliferativeeffects of the hBU12 and conjugates was observed. A lack of CDCanti-tumor effects was noticed for all hBU12 compounds. To determine therelevance of effector cell mediates activities for therapeutic activityof hBU12, human lymphoma and leukemia cells were implanted eithersubcutaneously into SCID mice or via tail vein injections (disseminatedmodel). The most potent anti-tumor effects were observed in disseminatedmodels, consistent with the notion that the access of effector cells tothe tumor cells is less limiting in the disseminated model (FIGS. 18 and19). In order to identify the nature of the cells mediating anti-tumoreffects, effector cell ablation experiments were conducted in adisseminated model of NHL (Ramos). NK cells, neutrophils or macrophageswere selectively depleted and the effects on tumor growth inhibition ofhBU12 in tumor bearing mice were measured. Ablation of macrophage andneutrophils almost completely abolished the anti-tumor effects of hBU12,while depletion of NK cells was associated with a moderate decrease inactivity. In conclusion, the findings demonstrate that hBU12 inducedanti-tumor effects via effector cell mediated ADCP and ADCC activities.

Material and Methods

For in vivo depletion studies, rabbit anti-asialo-GM-1 antibody wasobtained from Wako Pure Chemical Industries, Ltd. (Richmond, Va.), ratanti-mouse-Gr-1 antibody was obtained from BD Biosciences (San Diego,Calif.). Liposome-encapsulated clodronate (CEL) was prepared aspreviously described(Van Rooijen and Sanders 1994). Clodronate was agift of Roche Diagnostics GmbH (Mannheim, Germany). Tumor-bearing micewere depleted of effector cells using specific antibody or CEL asdescribed previously (Van Rooijen and Sanders 1994; van Rooijen andSanders 1997; McEarchern, Oflazoglu et al. 2007). Natural killer (NK)cells were depleted by i.p. injection of anti-asialo-GM 1 (1.25 mg/kg).Mice were given a total of 3 doses once every 5 days, beginning the dayof tumor cell implantation. Macrophages were depleted by i.p. injectionof CEL (100ρl/10 gr) on the day of tumor injection and every 3 daysthereafter for a total of 5 doses. Cell depletion was confirmed by flowcytometric analysis of splenocytes, lymph nodes and blood (data notshown).

SEQUENCE LISTING SEQ ID NO: 1: Met Gly Arg Leu Thr Ser Ser Phe Leu LeuLeu Ile Val Pro Ala Tyr Val Leu Ser SEQ ID NO: 2: (CDR regions areunderlined. Kabat positions 75, 79, 81, 82, 82A, 82B, 82C, and 89 are inbold font. Residues that determine CDR structure have an asterix* totheir right side) Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val LysPro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe* Ser Gly* Phe* Ser Leu* SerThr Ser Gly Met Gly Val* Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu GluTrp Leu Ala His Ile Trp Trp Asp Asp* Asp Lys Arg Tyr Asn Pro Ala Leu LysSer Arg Leu Thr Ile Ser Lys* Asp Thr Ser Lys Asn Gln Val Val Leu Thr MetThr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg*Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 3: Ala Ser Thr Lys Gly Pro Ser Val Phe Pro LeuAla Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu ValLys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu ThrSer Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile CysAsn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro LysSer Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr LysPro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn LysAla Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro ArgGlu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser ArgTrp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His AsnHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys SEQ ID NO: 4: (CDRregions are underlined. Kabat positions 75, 79, 81, 82, 82A, 82B, 82C,and 89 are in bold font) Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu ValLys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu SerThr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu GluTrp Leu AlaHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg LeuThr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val Val Leu Thr Met Thr Asn MetAsp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 5: (CDR regions are underlined. Kabat positions75, 79, 81, 82, 82A, 82B, 82C, and 89 are in bold font) Gln Val Thr LeuArg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr CysThr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile ArgGln Pro Pro Gly Lys Ala Leu Glu Trp Leu AlaHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg LeuThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Phe Leu Thr Met Thr Asn MetAsp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 6: (CDR regions are underlined. Kabat positions75, 79, 81, 82, 82A, 82B, 82C, and 89 are in bold font) Gln Val Thr LeuArg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr CysThr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile ArgGln Pro Pro Gly Lys Ala Leu Glu Trp Leu AlaHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg LeuThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Lys Ile Ala Ser ValAsp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 7: (CDR regions are underlined. Kabat positions75, 79, 81, 82, 82A, 82B, 82C, and 89 are in bold font) Gln Val Thr LeuArg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr CysThr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile ArgGln Pro Pro Gly Lys Ala Leu Glu Trp Leu AlaHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg LeuThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn MetAsp Pro Val Asp Thr Ala Ala Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 8: Gln Val Thr Leu Lys Glu Ser Gly Pro Gly IleLeu Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser LeuSer Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly LeuGlu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala LeuLys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val Phe Leu LysIle Ala Ser Val Asp Thr Ala Asp Thr Ala Ala Tyr Tyr Cys Ala Arg Met GluLeu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val SerSer SEQ ID NO: 9: (CDR regions are underlined. Kabat positions 24, 27,29, 71, 75, 78, 79, and 89 are in bold font. Residues that determine CDRstructure have an asterix* to their right side) Gln Val Gln Leu Gln GluSer Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val*Ser Gly* Gly* Ser Ile* Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Val* Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg*Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 10: (CDR regions are underlined. Kabat positions24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrVal Ser Gly Gly Ser Ile Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 11: (CDR regions are underlined. Kabat positions24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrPhe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 12: (CDR regions are underlined. Kabat positions24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrPhe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 13: (CDR regions are underlined. Kabat positions24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrVal Ser Gly Gly Ser Ile Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Val Asp Thr Ser Ser Asn Gln Phe Ser Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 14: (CDR regions are underlined. Kabat positions24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrVal Ser Gly Gly Ser Ile Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Val Asp Thr Ser Lys Asn Gln Val Phe Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 15: (CDR regions are underlined. Kabat positions24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrVal Ser Gly Gly Ser Ile Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg GlnHis Pro Gly Lys Gly Leu Glu Trp Ile GlyHis Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg ValThr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser ValThr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys Ala ArgMet Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val ThrVal Ser Ser SEQ ID NO: 16 Met Asp Phe Gln Val Gln Ile Phe Ser Phe LeuLeu Ile Ser Ala Ser Val Ile Met Ser Arg Gly SEQ ID NO: 17 (CDR regionsare underlined. Kabat positions 2, 40, 41, 42, 69, 70, 71, 72, and 83are in bold font. Residues that determine CDR structure have an asterix*to their right side) Glu Ile* Val Leu Thr Gln Ser Pro Ala Thr Leu SerLeu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe* Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe GlnGly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys ArgSEQ ID NO: 18 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser AspGlu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly AsnSer Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu SerSer Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg GlyGlu Cys SEQ ID NO: 19 (CDR regions are underlined. Kabat positions 2,40, 41, 42, 69, 70, 71, 72, and 83 are in bold font) Glu Asn Val Leu ThrGln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe GlnGly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys ArgSEQ ID NO: 20 (CDR regions are underlined. Kabat positions 2, 40, 41,42, 69, 70, 71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln SerPro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp His Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln GlySer Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQID NO: 21 (CDR regions are underlined. Kabat positions 2, 40, 41, 42,69, 70, 71, 72, and 83 are in bold font) Glu Asn Val Leu Thr Gln Ser ProAla Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp His Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe GlnGly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys ArgSEQ ID NO: 22 (CDR regions are underlined. Kabat positions 2, 40, 41,42, 69, 70, 71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln SerPro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser ThrAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly SerVal Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ IDNO: 23 (CDR regions are underlined. Kabat positions 2, 40, 41, 42, 69,70, 71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln Ser Pro AlaThr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Asn Ser His Phe Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln GlySer Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQID NO: 24 (CDR regions are underlined. Kabat positions 2, 40, 41, 42,69, 70, 71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln Ser ProAla Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser LeuGlu Pro Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln GlySer Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQID NO: 25 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser ProGly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met HisTrp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr Asp Thr SerLys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn SerHis Phe Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Val Ala Thr Tyr Tyr CysPhe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu IleLys Arg SEQ ID NO: 26 (CDR regions are underlined. Kabat positions 2,and 71 are in bold font. Residues that determine CDR structure have anasterix* to their right side) Glu Ile* Val Leu Thr Gln Ser Pro Asp PheGln Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Asp GlnSer Pro Lys Leu Leu Ile Lys Asp Thr Ser Lys Leu Ala Ser Gly Val Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe* Thr Leu Thr Ile Asn Ser LeuGlu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly SerVal Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ IDNO: 27 (CDR regions are underlined. Kabat positions 2, and 71 are inbold font) Glu Asn Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr ProLys Glu Lys Val Thr Ile Thr CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Asp GlnSer Pro Lys Leu Leu Ile Lys Asp Thr Ser Lys Leu Ala Ser Gly Val Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp His Thr Leu Thr Ile Asn Ser LeuGlu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly SerVal Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ IDNO: 28 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr GlnThr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly MetGly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala HisIle Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Leu ThrIle Ser Lys Asp Thr Ser Xa Asn Gln Val Xb Leu Xc Xd Xe Xf Xg Asp Pro ValAsp Thr Ala Xh Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe AspTyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser wherein Xa is Ser orLys, Xb is Phe or Val, Xc is Lys or Thr, Xd is Ile or Met, Xe is Ala orThr Xf is Ser or Asn, Xg is Val or Met, and Xh is Ala or Thr. SEQ ID NO:29 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln ThrLeu Ser Leu Thr Cys Thr Xa Ser Gly Xb Ser Xc Ser Thr Ser Gly Met Gly ValGly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly His Ile TrpTrp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Val Thr Ile SerXd Asp Thr Ser Xe Asn Gln Xf Xg Leu Lys Leu Ser Ser Val Thr Ala Ala AspThr Ala Xh Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp TyrTrp Gly Gln Gly Thr Leu Val Thr Val Ser Ser wherein Xa is Phe or Val, Xbis Phe or Gly, Xc is Leu or Ile, Xd is Lys or Val, Xe is Ser or Lys, Xfis Val or Phe, Xg is Phe or Ser, and Xh is ala or Val. SEQ ID NO: 30 GluXa Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg AlaThr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln GlnLys Xb Xc Xd Ala Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser GlyIle Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Xe Xf Xg Xh Leu Thr Ile SerSer Leu Glu Pro Glu Asp Xi Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val TyrPro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg wherein Xa isAsn or Ile, Xb is Ser or Pro, Xc is Ser or Gly, Xd is Thr or Gln, Xe isAsn or Thr, Xf is Ser or Asp, Xg is His or Phe, Xh is Phe or Thr, and Xiis Val or Phe. SEQ ID NO: 31 Glu Xa Val Leu Thr Gln Ser Pro Asp Phe GlnSer Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser ValSer Tyr Met His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu IleLys Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser GlySer Gly Thr Asp Xb Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala AlaThr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly ThrLys Leu Glu Ile Lys Arg wherein Xa is Asn or Ile and Xb is His or Phe.SEQ ID NO: 32 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys ProThr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr SerGly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp LeuAla His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser ArgLeu Thr Ile Ser Lys Asp Thr Ser Xa Asn Gln Val Xb Leu Xc Xd Xe Xf Xg AspPro Val Asp Thr Ala Xh Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr TyrPhe Asp Tyr Trp Gly Xi Gly Thr Xj Val Thr Val Ser Ser wherein Xa is Seror Lys, Xb is Phe or Val, Xc is Lys or Thr, Xd is Ile or Met, Xe is Alaor Thr Xf is Ser or Asn, Xg is Val or Met, Xh is Ala or Thr, Xi is Glnor Arg, and Xj is Leu, Thr, or Met. SEQ ID NO: 33 Gln Val Gln Leu GlnGlu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys ThrXa Ser Gly Xb Ser Xc Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln HisPro Gly Lys Gly Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys ArgTyr Asn Pro Ala Leu Lys Ser Arg Val Thr Ile Ser Xd Asp Thr Ser Xe AsnGln Xf Xg Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xh Tyr Tyr CysAla Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Xi Gly Thr XjVal Thr Val Ser Ser wherein Xa is Phe or Val, Xb is Phe or Gly, Xc isLeu or Ile, Xd is Lys or Val, Xe is Ser or Lys, Xf is Val or Phe, Xg isPhe or Ser, Xh is Ala or Val, Xi is Gln or Arg, and Xj is Leu, Thr, orMet. SEQ ID NO: 34 Glu Xa Val Leu Thr Gln Ser Pro Ala Thr Leu Ser LeuSer Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser TyrMet His Trp Tyr Gln Gln Lys Xb Xc Xd Ala Pro Arg Leu Leu Ile Tyr Asp ThrSer Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly XeXf Xg Xh Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Xi Ala Val Tyr Tyr CysPhe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Xj Gly Thr Xk Xl Xm Ile LysArg wherein Xa is Asn or Ile, Xb is Ser or Pro, Xc is Ser or Gly, Xd isThr or Gln, Xe is Asn or Thr, Xf is Ser or Asp, Xg is His or Phe, Xh isPhe or Thr, Xi is Val or Phe, Xj is Gln, Pro or Gly, Xk is Lys or Arg,XI is Leu or Val, and Xm is Glu or Asp. SEQ ID NO: 35 Glu Xa Val Leu ThrGln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr CysSer Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Asp GlnSer Pro Lys Leu Leu Ile Lys Asp Thr Ser Lys Leu Ala Ser Gly Val Pro SerArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Xb Thr Leu Thr Ile Asn Ser LeuGlu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro PheThr Phe Gly Xc Gly Thr Xd Xe Xf Ile Lys Arg wherein Xa is Asn or Ile, Xbis His or Phe, Xc is Gln, Pro or Gly, Xd is Lys or Arg, Xe is Leu orVal, and Xf is Glu or Asp. SEQ ID NO: 36ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 37QMQGVNCTVSSELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVQVHNAKTKPREQQFNSTFRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK SEQ ID NO: 38ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 39ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 40atgggcaggcttacttcttcattcttgttgctgattgtccctgcatatgtcctgtcc SEQ ID NO: 41caggttcagctgcaagagtctggccctgggttggttaagccctcccagaccctcagtctgacttgtactgtgtctgggggttcaatcagcacttctggtatgggtgtaggctggattaggcagcacccagggaagggtctggagtggattggacacatttggtgggatgatgacaagagatataacccagccctgaagagcagagtgacaatctctgtggatacctccaagaaccagtttagcctcaagctgtccagtgtgacagctgcagatactgctgtctactactgtgctagaatggaactttggtcctactattttgactactggggccaaggcacccttgtcacagtctcctca SEQ ID NO: 42gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaSEQ ID NO: 43atggattttcaagtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtccagaggagaaaSEQ ID NO: 44ttgttctcacccagtctccagcaaccctgtctctctctccaggggaaagggctaccctgagctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagccagggcaggctcccagactcctgatttatgacacatccaaactggcttctggtattccagcaaggttcagtggcagtgggtctggaacagattttacactcacaatcagcagcctggagccagaggatgttgctgtctattactgttttcaggggagtgtatacccattcacttttggccaagggacaaagttggaaatcaaaaga SEQ ID NO: 45actgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ ID NO: 46 Thr Ser Gly Met Gly Val Gly SEQ ID NO: 47His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser SEQ IDNO: 48 Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr SEQ ID NO: 49 Ser Ala SerSer Ser Val Ser Tyr Met His SEQ ID NO: 50 Asp Thr Ser Lys Leu Ala SerSEQ ID NO: 51 Phe Gln Gly Ser Val Tyr Pro Phe Thr

1-15. (canceled)
 16. A CD19 binding agent that specifically binds tohuman CD19, said binding agent comprising a heavy chain variable regioncomprising an amino acid sequence at least 90% identical to SEQ ID NO:2;the binding agent binding to human CD19 with a dissociation constant ofless than 10⁻⁷M.
 17. The CD19 binding agent of claim 16 thatspecifically binds to human CD19 and comprises a heavy chain variableregion comprising an amino acid sequence at least 90% identical to SEQID NO:2; the binding agent binding to human CD19 with a dissociationconstant of less than 10⁻⁷M and comprising CDR regions having the aminoacid sequences set forth in SEQ ID NOS. 46, 47, and
 48. 18. The bindingagent of claim 16 further comprising a light chain variable regioncomprising an amino acid sequence at least 90% identical to SEQ ID NO:17or SEQ ID NO:26.
 19. The CD19 binding agent of claim 16, furthercomprising a human IgG constant region joined to the heavy chainvariable region.
 20. The CD19 binding agent of claim 19, wherein theisotype of IgG constant region is IgG1, IgG2, or IgG1V1.
 21. The CD19binding agent of claim 18 further comprising a light chain constantdomain joined to the light chain variable region, wherein the lightchain constant domain is a kappa constant domain.
 22. The CD19 bindingagent of claim 16 that comprises a humanized antibody.
 23. The CD19binding agent of claim 16 wherein the CD19 binding agent is conjugatedto a cytotoxic agent.
 24. A ligand-drug conjugate compound of thefollowing formula:L-(LU-D)_(p)  (I) or a pharmaceutically acceptable salt or solvatethereof; wherein: L is a ligand unit wherein the ligand unit is a CD19binding agent of claim 16; and (LU-D) is a Linker unit-Drug unit moiety,wherein: LU- is a Linker unit, and -D is a Drug unit having cytostaticor cytotoxic activity against the target cells; and p is an integer from1 to about
 20. 25. The ligand-drug conjugate compound of claim 24,wherein the ligand-drug conjugate compound has the formula:

or a pharmaceutically acceptable salt or solvate form thereof, whereinmAb-S— is a CD19 binding agent of claim 1 and p is from 1 to
 8. 26. Aligand-drug conjugate compound having the formula:

or a pharmaceutically acceptable salt or solvate form thereof; whereinmAb-S— is a CD19 binding agent comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:9 and comprising a lightchain variable region comprising the amino acid sequence of SEQ ID NO:24and p is from 1 to
 8. or a pharmaceutically acceptable salt or solvateform thereof.
 27. A pharmaceutical composition comprising theligand-drug conjugate compound of claim 1, and a pharmaceuticallyacceptable carrier or excipient.
 28. A method for treating aCD19-associated disorder in a mammalian subject comprising administeringa pharmaceutical composition of claim 27 in an amount effective to treatthe disorder in the mammalian subject.
 29. The method of claim 28wherein the CD19-associated disorder is a CD19 expressing cancer.
 30. Amethod for treating a subject that has a cancer that is refractory totreatment with rituximab comprising administering to the subject apharmaceutical composition of claim 27 in an amount effective to treatthe cancer.
 31. A method of manufacturing a ligand-drug conjugatecompound comprising conjugating a CD19 binding agent of claim 1 to acytotoxic agent.
 32. A method of manufacturing a ligand-drug conjugatecompound comprising conjugating a CD19 binding agent of claim 1 to alinker unit conjugated to a drug unit.
 33. The binding agent of claim 3comprising a humanized antibody wherein the heavy chain variable regioncomprises an amino acid sequence at least 90% identical to SEQ ID NO:2and the light chain variable region comprises an amino acid sequence atleast 90% identical to SEQ ID NO:17.
 34. The humanized antibody of claim33, wherein any heavy chain variable region framework positions thatdiffer from SEQ ID NO:2 are occupied by the amino acids occupying thecorresponding positions of SEQ ID NO:8 or SEQ ID NO:32; and wherein anylight chain variable region framework positions that differ from SEQ IDNO:17 are occupied by the amino acids occupying the correspondingposition of SEQ ID NO:25 or SEQ ID NO:34.
 35. The humanized antibody ofclaim 34, wherein any heavy chain variable region framework positionsthat differ from SEQ ID NO:2 and the amino acids occupying thosepositions are selected from the group consisting of positions H75, H79,H81, H82, H82A, H82B, H82C, and H89 occupied by S, F, K, I, A, S, V, andA respectively; and any light chain variable region framework positionsthat differ from SEQ ID NO.17 and the amino acids occupying thosepositions are selected from the group consisting of positions L2, L40,L41, L42, L69, L70, L71, L72 and L83 occupied by N, S, S, T, N, S, H, F,and V respectively.
 36. The humanized antibody of claim 35, wherein thelight chain variable region framework position L83 is occupied by V. 37.The humanized antibody of claim 33, wherein at least one of the heavychain variable region positions H75, H79, H81, H82, H82A, H82B, H82C andH89 is occupied by S, F, K, I, A, S, V, and A respectively.
 38. Thehumanized antibody of claim 33, wherein at least one of the light chainvariable region positions L2, L69, L71, L72 and L83 is occupied by N, N,H, F and V respectively.
 39. The humanized antibody of claim 33, whereinno more than three amino acids in the heavy chain variable regionframework differ from SEQ ID NO:2, and no more than three amino acids inthe light chain variable region framework differ from SEQ ID NO:17.